Mouse embryonic fibroblasts (MEFs) (Karbowski et al., 2006), mouse pituitary αT3 cells and monkey kidney Cos-7 cells were maintained at 37°C under 5% CO₂ in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Human embryonic kidney (HEK293T), human colon carcinoma (HCT116) (Wang and Youle, 2012), and human HeLa cells were maintained identically, except with 10% serum. Rabbit antibodies used were: anti-IP₃R1, anti-IP₃R2 (Wojcikiewicz, 1995), anti-erlin2, anti-gp78, anti-Hrd1 (Pearce et al., 2007), anti-Mcl-1 #D35A5, anti-Bcl-2 #D17C4, anti-caspase-3 #9662, anti-Bak #12105, anti-Bax #2772, anti-LC3A/B #4108, anti-PARP #9542, anti-caspase-8 #8592 (Cell Signaling Technology), anti-Bok^A (raised against amino acids 19–32 of mouse Bok) (Ke et al., 2012; Echeverry et al., 2013), anti-Bok^B (Abcam # 186745, raised against amino acids 1–150 of human Bok), and anti-transaldolase (Pearce et al., 2007). Mouse monoclonal antibodies used were: anti-CHOP #7351, anti-ube2J1 #100624 (Santa Cruz Biotechnology Inc.), anti-ubiquitin clone FK2 (BioMol), anti-FLAG epitope clone M2 #F3165 (Sigma), anti-HA epitope clone HA11 (Covance), anti-IP₃R3 #610313 (BD Transduction Labs), anti-p97 #10R-P104A (Fitzgerald), anti-cytochrome c #13575 and anti-complex V/III #14748/14745 (Abcam). Horseradish peroxidase-conjugated secondary antibodies, protease inhibitors, Triton X-100, CHAPS, brefeldin A (BFA), tunicamycin (TN), thapsigargin (TG), bafilomycinA1 (BAFA1), and cycloheximide (CHX) were purchased from Sigma. MG132 and staurosporine (SST) were from Enzo Life Sciences. Q-VD-OPH and CB-5083 were from Cayman Chemical. Protein A-Sepharose CL-4B was from GE Healthcare. Linear, MW∼25,000 polyethylenimine (PEI) was from Polysciences Inc. Precision Plus™ Protein Standards, and SDS-PAGE reagents were from Bio-Rad. TAK-243 was from MedChemExpress. Lipofectamine 2000 and Opti-MEM were from ThermoFisher. Trypsin-EDTA and Trypan Blue were from Corning.

Cell lysates and IPs were prepared as described (Schulman et al., 2019). Mouse brain, rat brain, and human kidney (obtained from the Upstate Biorepository Center) were prepared as described (Schulman et al., 2016). All samples were resuspended in gel-loading buffer (Oberdorf et al., 1999), incubated at 37°C for 30 min, subjected to SDS-PAGE, and proteins were transferred to nitrocellulose for probing as described (Schulman et al., 2019). Immunoreactivity was detected using Pico Chemiluminescent Substrates (ThermoFisher #34579) and a ChemiDoc imager (Bio-Rad).

The CRISPR/Cas9 system was used to target exons within the Bok, IP₃R1, IP₃R2, ube2J1, gp78, erlin2, and Hrd1 genes using the gRNAs indicated (Supplementary Table S1), with constructs introduced by Neon transfection (Invitrogen; 10 µg total DNA per 3 × 10⁶ cells in 100 μl, 1 pulse, 20 ms, 1,500 V). As previously described (Schulman et al., 2013), IP₃R1 was targeted in MEFs using either gRNA 1 or 2 together with vectors encoding hCas9 (Addgene) and enhanced green fluorescent protein (EGFP) (Clontech). IP₃R2 was then targeted in wild-type and IP₃R1 KO MEFs using gRNA 1 or 2 in the pCas-Guide-EF1a-GFP vector (Origene) to generate IP₃R2 KO and IP₃R1/2 KO MEFs. Bok was targeted using gRNA 1 or 2 (Schulman et al., 2019) in wild-type and Bak/Bax double KO MEFs (DKO MEFs) (Karbowski et al., 2006) together with vectors encoding hCas9 and the Clover EGFP mutant (Addgene) to generate Bok KO MEFs (BKO MEFs) and Bak/Bax/Bok triple KO MEFs (TKO MEFs). Ube2J1, gp78, erlin2, and Hrd1 were targeted in IP₃R1/2 KO MEFs using gRNAs in the pCas-Guide-EF1a-GFP vector (Origene). In all cases, fluorescent protein-expressing cells were isolated 48 h post-transfection by fluorescence-activated cell sorting (FACS) and plated at 1 cell/well in 96-well plates. Colonies were expanded and screened in immunoblots for loss of target protein. Multiple independent cell lines from each gRNA were used for all experiments. In some experiments (Figure 2B), sorted cells were plated at 10,000 cells/9.6 cm² well, which were expanded and analyzed as “heterogenous cultures.”

Mouse Bok tagged at the N-terminus with a triple FLAG epitope (3F-Bok) (Schulman et al., 2016) was used to generate 3F-Bok^6KR, 3F-Bok^K160R, and 3F-Bok^K160A by mutagenic PCR. HEK293T and Bak/Bax KO double (DKO) HCT116 cells (Wang and Youle, 2012) seeded at 6 × 10⁵/9.6 cm² well were transfected ∼24 h later with 0.125 µg of Bok cDNAs and 6 µl of 1 mg/ml PEI (pre-mixed in 50 µl of serum-free cultured medium), and ∼24 h later were harvested with ∼0.2 ml/well lysis buffer and analyzed via immunoblotting. Mouse 3F-Bok^R200A/K203A and 3F-Bok^Δ™ (amino acids 1–187 of mouse Bok) were generated as described (Schulman et al., 2016; Szczesniak et al., 2021a). MEFs transfected with 3F-Bok constructs and mouse IP₃R1HA (Szczesniak et al., 2021a; Gao et al., 2022) using the Neon Transfection system described above were seeded at 6 × 10⁵/9.6 cm² well and 48 h post-transfection, cells were harvested with ∼0.1 ml/well lysis buffer. HeLa Bok KO cells seeded at 3 × 10⁵/9.6 cm² well were transfected with mouse 1F-Mcl-1, 3F-Bok^WT, 3F-Bok^R200A/K203A or 3F-Bok^Δ™ using 5 µl of Lipofectamine 2000 and ∼24 h later were harvested with ∼0.1 ml/well lysis buffer. The authenticity of all cDNAs was confirmed by DNA sequencing (Genewiz).

Cells were seeded at 3 × 10⁵/9.6 cm² well and were transfected with 3F-Bok^WT or 3F-Bok^R200A/K203A, or were treated with 10 µM of MG132 for various times. Cells were then harvested with 0.5 ml trypsin-EDTA and then mixed with trypan blue at a final concentration of 0.2% w/v for 10 min. Cells were counted with a TC20 Automated Cell Counter (Bio-Rad) and cell death was measured by the inability to exclude trypan blue.

As previously described (Wright et al., 2018), αT3 IP₃R1 KO cells (Schulman et al., 2016) were transfected to express mouse IP₃R1HA^WT and IP₃R1HA^{Δ 1916/17} using the Neon Transfection System followed by selection in 1.3 mg/ml G418 for 72 h. Cells were then plated at 1 cell/well in 96-well plates, expanded, screened in immunoblots for IP₃R1 expression, and maintained in 0.3 mg/ml G418. Multiple clones were characterized in all experiments.

RNA was extracted from 10⁶ MEFs using the RNeasy Mini Kit (Qiagen) and reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). qRT-PCR for Bok mRNA levels was performed using the forward primer GAT​GGA​CGG​ATG​TCC​TCA​AG and the reverse primer TCT​CTG​GCA​ACA​ACA​GGA​AG (Schulman et al., 2016) with SsoAdvanced Universal SYBR^® Green Supermix, a CFX Connect Real-Time PCR Detection System thermal cycler (Bio-Rad), and the following PCR cycling parameters: 30 s at 95°C and then 40 cycles of 95°C for 15 s and 60°C for 30 s. Results were analyzed by normalizing to the housekeeping genes S18, peptidylpropyl isomerase A, and hypoxanthine phosphoribosyl-transferase 1, using the Bio-Rad CFX Maestro Software and the 2^−ΔΔCT method (Schulman et al., 2016).

Cell fractionation was performed using WT and IP₃R1/2 KO MEFs essentially as described (Wieckowski et al., 2009; Morciano et al., 2016) with all steps at 4°C. Approximately 10⁸ cells were collected in 155 mM NaCl, 10 mM HEPES, 1 mM EDTA, pH 7.4, and centrifuged at 500 × g for 5 min. Cell pellets were then washed with phosphate-buffered saline and re-centrifuged at 500 × g for 5 min. Cell pellets were then resuspended with 10 ml homogenization buffer (225 mM mannitol, 75 mM sucrose, 30 mM Tris-HCl, 0.1 mM EGTA, 10 μM pepstatin, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 0.2 μM soybean trypsin inhibitor, pH 7.4) and disrupted using a Kinematica Polytron^® PT 3100 Homogenizer for 5–10 s to obtain ∼70% plasma membrane disruption as indicated by trypan blue. The homogenized cells were centrifuged at 600 × g for 5 min to remove nuclei and unbroken cells. The supernatant (Homogenate, H) was then centrifuged at 1,800 × g for 10 min to pellet the crude mitochondria (CM). The subsequent supernatant was then centrifuged at 100,000 × g for 90 min using a Beckman Optima™ L-90K Ultracentrifuge (70-Ti rotor) to isolate the ER (pellet) and the cytosol, C (supernatant). The CM and ER pellets were resuspended in 10 ml homogenization buffer and subjected to SDS-PAGE along with H and C fractions.

All experiments were repeated two or more times (n = the number of independent experiments) and representative images of immunoblots and traces are shown. Quantitated data are expressed as mean ± SEM. Immunoreactivity quantification was performed using ImageLab software (BioRad). The appropriate bands were highlighted and volume intensity (high signal/noise ratio) or adjusted volume intensity (low signal/noise ratio) was measured in arbitrary units, A.U. Statistical analysis was performed using a Student’s t-test (with Welch’s correction in Figure 7) and p-values of <0.05 and <0.005 were considered statistically significant and denoted with “*” and “**” respectively, while p-values of >0.05 were not considered statistically significant and denoted with “#”.

It has been reported that Bok expression in cell lines is highly variable (e.g., undetectable in MEFs, but relatively high in HCT116 cells), and that Bok expression is strongly enhanced by proteasome inhibition (Llambi et al., 2016). Since this has not been universally observed and indeed, proteasome inhibition often does not affect Bok levels (Echeverry et al., 2013; Schulman et al., 2013; Carpio et al., 2015; D'Orsi et al., 2016; Schulman et al., 2016; Moravcikova et al., 2017; Schulman et al., 2019), we sought to clarify the situation by re-examining Bok levels in various key cell lines with multiple Bok antibodies. We find that Bok is readily-detectable at 23/21 kDa in MEFs; both in WT MEFs and in MEFs lacking Bak/Bax (double knockout “DKO” MEFs), with both anti-Bok^A and anti-Bok^B (Figure 1A, lanes 2 and 3). Lack of immunoreactivity in Bok KO MEFs (Schulman et al., 2019) served as a negative control for the antibodies, showing that they specifically recognize Bok (Figure 1A, lane 1). We have shown previously that other mouse cell lines and tissues express 23/21 kDa Bok, and that the two species result from the use of alternative translation start sites at codons for Met1 and Met15 (Schulman et al., 2016). Likewise, identical to mouse tissue (Figure 1A, lane 4), rat tissue expresses 23/21 kDa Bok (lane 5). Readily-detectable levels of Bok immunoreactivity were also seen in WT HCT116 cells and in HCT116 cells lacking Bak/Bax (DKO HCT116 cells), although therein Bok migrated predominantly at 22 kDa (lanes 6 and 7). Bok also migrates predominantly at 22 kDa in human tissue and monkey Cos-7 cells (with also a weak immunoreactive band at 20 kDa), and in HEK293T cells, in which Bok immunoreactivity was particularly low (lanes 8–10). The discrepancy between the apparent size of mouse and human Bok was also seen when they were expressed from cDNAs in HEK293T cells; exogenous mouse and human Bok migrated at 23/21 kDa and 22/20 kDa, respectively (lanes 11 and 12), indicating that both have the capacity to utilize alternative translation start sites. The reason for the migration discrepancy is surprising given that the mouse/rat versus human/monkey amino acid sequences are 95% identical (Figure 1B), but is presumably accounted for by slight sequence variations (e.g., glycine vs. no glycine at position 62 and proline vs. serine at position 96).

Also, in contrast to previous findings (Llambi et al., 2016), we observed that proteasomal inhibition did not alter endogenous Bok levels, since Bok immunoreactivity measured with both anti-Bok^A and anti-Bok^B was largely unaltered by treatment of cells with the proteasome inhibitor MG132, in the absence or presence of the pan-caspase inhibitor Q-VD-OPh, which should protect cells against potential apoptosis if Bok was elevated (Figure 1C). Further, we did not observe an increase in the expression level of exogenous Bok when all of the six lysine residues that could potentially serve as ubiquitination sites (Figure 1B) were blocked by mutation to arginine (Figure 1D). In fact, and very surprisingly, the lysine-free Bok mutant was partially fragmented (Figure 1D, lanes 3 and 6), for a reason that can be attributed to mutation of K160 to arginine (Supplementary Figure S1). While the reason for the discrepancy between our findings and those previously reported (Llambi et al., 2016) are unclear, the notion that cell survival is dependent upon constitutive ubiquitination and proteasomal degradation of Bok to vanishingly low levels appears to require re-evaluation.

Because Bok is well-expressed under normal conditions (Figure 1A), we examined whether it might indeed play a role in mediating the pro-apoptotic effects of proteasome inhibitors. We have shown previously (Schulman et al., 2019), as have others (Fernandez-Marrero et al., 2016), that deletion of just Bok from MEFs does not alter responsiveness to various triggers of apoptosis, including SST, MG132, TG, BFA and TN, suggesting that in MEFs, Bok is not a primary mediator of apoptosis, but leaving open the possibility that it might act redundantly with the bona fide MOMP mediators, Bak and Bax (Ke et al., 2015; Ke et al., 2018). Thus, we explored the role of Bok in Bak/Bax double (DKO) MEFs, since in these cells, it might be possible to clearly identify an apoptotic role for Bok. Interestingly, initial examination of the effects of the various triggers of apoptosis in WT or DKO MEFs (as indicated by caspase-3 cleavage) revealed that only MG132 retained activity in DKO MEFs (Figure 2A, lane 6), suggesting that Bok could potentially be contributing to the pro-apoptotic effects of MG132. However, MG132-induced caspase-3 cleavage was essentially identical in DKO and Bak/Bax/Bok triple KO (TKO) MEFs, either when examined shortly after Bok deletion in heterogeneous cultures (Figure 2B, lanes 6 vs. 8), or after the creation of clonal cell lines (Figure 2C, lanes 2 vs. 4). Cell death measurements (Figure 2D) showed that the effects of 4 h with MG132 were negligible (as expected, considering the relatively low amount of caspase-3 cleavage at that time point, Figure 2A), and that even the increased cell death seen at longer times with MG132 was still unaffected by Bok deletion. Thus, in MEFs, MG132 effects are largely Bak/Bax/Bok independent, and clearly, Bok does not mediate proteasome inhibitor-induced apoptosis.

With regard to the mechanism of MG132 action, caspase-8 cleavage was enhanced by MG132 in parallel to caspase-3 (Figures 2B,C), suggesting that MG132 may be triggering activation of the death receptor or “extrinsic” apoptosis pathway, which in turn causes caspase-3 cleavage (Tummers and Green, 2017; Carneiro and El-Deiry, 2020). Possible MOMP involvement was also assessed by measuring cytochrome c release from mitochondria, but revealed no substantial effect of MG132 (Supplementary Figure S2). Thus, MG132-induced apoptosis in MEFs appears to occur independently of MOMP, consistent with other studies (Laussmann et al., 2011; Hellwig et al., 2022).

Since it has been shown previously that the stability and expression level of endogenous Bok are governed by binding to IP₃Rs in αT3 cells and DT40 cells (Schulman et al., 2016; Schulman et al., 2019), we wondered if the same is true in MEFs, in which endogenous Bok is clearly expressed well and is not actively degraded by the proteasome (Figure 1C). Bok binds strongly to IP₃R1 and IP₃R2, but not to IP₃R3, and since MEFs contain IP₃R1-3 (Schulman et al., 2013) we individually and then consecutively targeted IP₃R1 and IP₃R2 using CRISPR/Cas9-mediated gene editing, and specifically deleted these proteins (Figure 3A). Remarkably, deletion of IP₃R1 caused a major decline in Bok levels (Figure 3A, lanes 3 and 4), while deletion of IP₃R2 had little or no effect (lanes 5 and 6), consistent with the relatively small contribution of IP₃R2 to total MEF IP₃R content (Schulman et al., 2013). Deletion of both IP₃R1 and IP₃R2 caused the most dramatic decline in Bok levels (lanes 7 and 8). Levels of Bcl-2 were unaffected, showing that the effect is specific to Bok. The expression level of Bok in IP₃R1/2 KO MEFs was ∼2% of that seen in control cells, since the Bok immunoreactivity seen with 50 µg of IP₃R1/2 KO cell lysate was approximately equivalent to that seen with 1 µg of WT cell lysate (Supplementary Figure S3A). The decline in Bok expression was apparently not due to a change in Bok synthesis, as Bok mRNA levels were not substantially different between WT and IP₃R KO cell lines (Figure 3A, histogram), indicating that Bok is degraded when IP₃R1 and IP₃R2 are absent. The proteasome appears to be the degradation route, since treatment with MG132 caused a rapid increase in Bok levels in IP₃R1/2 KO cells (Figure 3B, lanes 7–12), that was not seen in WT cells (lanes 1–6). In IP₃R1/2 KO cells, Bok immunoreactivity increased ∼5-fold after 1 h with MG132 (lane 7 vs. 10), to a level ∼10% of that seen in WT cells (Supplementary Figure S3B). Further, 1 h with MG132 did not significantly alter Bok mRNA levels in IP₃R1/2 KO MEFs (levels after MG132 treatment were 119 ± 15% of that in control cells; mean ± n = 5). Thus, in IP₃R1/2 KO cells, MG132 markedly increases Bok immunoreactivity without affecting Bok mRNA levels, suggesting that newly-synthesized Bok is degraded by the proteasome when it is unable to access IP₃R binding sites. Other classes of UPP blocking drugs also elevated Bok levels; the p97 inhibitor CB-5083 (Anderson et al., 2015) and the UBE1 inhibitor TAK-243 (Hyer et al., 2018) both increased Bok levels similarly to MG132 (Figure 3C, lanes 2–4). In contrast, the lysosome inhibitor bafilomycin A1 did not affect Bok levels (lane 5). Why Bok levels in IP₃R1/2 KO cells are only increased to ∼10% of WT levels by UPP inhibition is presently unclear, but is most likely because UPP blockade inhibits protein synthesis (Guan et al., 2014) thus limiting Bok restoration.

Regarding the enzymes/proteins that could be mediating Bok degradation in IP₃R1/2 KO MEFs, we examined several candidates (ube2J1, gp78, Hrd1, and erlin2) (Christianson and Carvalho, 2022) via CRISPR/Cas9-mediated deletion. However, none of these deletions increased Bok immunoreactivity, indicating these proteins are not involved (Supplementary Figure S4).

Exogenous IP₃R1HA constructs (Szczesniak et al., 2021a; Gao et al., 2022) were expressed transiently in IP₃R1/2 KO MEFs (Figure 4A). IP₃R1HA^WT increased endogenous Bok levels, indicating that if normal Bok binding sites are introduced, newly synthesized Bok is stabilized (lane 2 vs. 1). The specificity of this effect is demonstrated by the finding that IP₃R1HA^{Δ 1916/17}, which does not bind Bok (Szczesniak et al., 2021a), did not restore Bok levels (lane 3). Likewise, IP₃R1HA^Δ™, which lacks TM domains 1–6 and localizes to the cytosol (Szczesniak et al., 2021a), did not restore Bok expression, indicating that events that control Bok stability occur at the ER membrane (lane 4). Finally, IP₃R1HA^Δ1-223, which lacks the “suppressor domain” and has no Ca²⁺ channel activity (Uchida et al., 2003), also restored Bok levels, indicating that it is the presence of ER-located Bok binding sites, rather than functional Ca²⁺ channels that stabilizes Bok (lane 5). Similar observations can be made in IP₃R1 KO αT3 cells (Schulman et al., 2016) in which stable expression of exogenous IP₃R1HA^WT, but not IP₃R1HA^{Δ 1916/17}, partially restores endogenous Bok expression (Figure 4B). The lack of Bok restoration with IP₃R1HA^{Δ 1916/17} is not because of impaired functionality, since IP₃R1HA^{Δ 1916/17} restores GnRH- induced Ca²⁺ mobilization in IP₃R1 KO αT3 cells just as well as IP₃R1HA^WT (Supplementary Figure S5). Overall, these data indicate that Bok is stabilized by its binding site present in membrane-bound IP₃Rs, but without any need for IP₃R channel activity. These conclusions were confirmed in a different cell line, IP₃R1-3 KO HeLa cells (Ando et al., 2018), which express much less Bok than unmodified HeLa cells, and in which TAK-243 and IP₃R1HA^WT (but not IP₃R1HA^{Δ 1916/17}) partially restore endogenous Bok levels (Supplementary Figure S6).

To better understand the route of Bok degradation in IP₃R1/2 KO MEFs, subcellular fractionation was performed (Figure 5A). As expected, in WT MEFs (Figure 5B, lanes 1–4), Bok was found predominantly in the ER fraction with none in the cytosol (C) fraction, but with also some in the crude mitochondria (CM) fraction; ER:CM ratio ∼68:32 (Figure 5C). The presence of some ER in the CM fraction, as indicated by IP₃R1 and IP₃R3, likely accounts for the presence of Bok in the CM fraction. The residual Bok present in IP₃R1/2 KO MEFs was not detectable using subcellular fractionation (Figure 5B, lanes 5–8), however, TAK-243 treatment revealed that stabilized Bok was predominately in the ER fraction with none in C fraction (Figure 5B, lanes 9–12) and with approximately the same distribution as that seen in WT cells; ER:CM ratio ∼61:39 (Figure 5C). Thus, in the presence or absence of Bok binding sites provided by IP₃Rs, Bok is inserted into the ER membrane.

A feature of Bok unique among Bcl-2 family proteins is the presence of two charged residues (R200 and K203) within the TM domain (Figure 6A). To explore their significance, these two residues were mutated to alanine, creating 3F-Bok^R200A/K203A (Figure 6A). Analysis in comparison to 3F-Bok^WT in transiently transfected IP₃R1/2 KO MEFs showed that the expression of both constructs was greatly enhanced by co-expression of IP₃R1HA^WT, indicating that both are localized to the ER membrane and stabilized by IP₃R1 binding (Figure 6B). Further examination of stability using cycloheximide (CHX) chase showed that both constructs were turned-over very slowly in the presence of IP₃R1HA^WT (Figures 6C,E), while in the absence of IP₃R1HA^WT, the constructs were turned-over rapidly, with 3F-Bok^R200A/K203A being slightly less stable than 3F-Bok^WT (Figures 6D,E). Thus, while R200 and K203 do not affect the ability of Bok to interact with and be stabilized by IP₃Rs, they slightly influence the stability of non-IP₃R-bound “free” Bok.

To investigate how R200 and K203 might impact the ability of over-expressed Bok to trigger apoptosis, we utilized Bok KO HeLa cells (Szczesniak et al., 2021b), since in this cell type exogenous Bok can induce apoptosis, most likely due to high transfection efficiency and high Bok over-expression levels (Schulman et al., 2016; Chernousova and Epple, 2017; Szczesniak et al., 2021b). Intriguingly, 3F-Bok^R200A/K203A caused significantly more apoptosis than 3F-Bok^WT, as indicated by caspase-3 and PARP cleavage, as well as cell death (Figure 7A, lanes 1–3, blot, histogram and insert). As exogenous Bok and anti-apoptotic Mcl-1 interact (Lucendo et al., 2020; Szczesniak et al., 2021b), we examined the effects of Mcl-1 co-expression and found that Mcl-1 blocked the apoptotic effects of both 3F-Bok^WT and 3F-Bok^R200A/K203A while, surprisingly, strongly enhancing their expression (Figure 7A, lanes 4–6, blot and histogram). Consistent with these findings, both constructs co-immunoprecipitated with exogenous Mcl-1, with 3F-Bok^R200A/K203A interacting slightly more strongly than did 3F-Bok^WT (Figure 7B, lane 4 vs. 2). These data suggest that 3F-Bok^R200A/K203A may exhibit enhanced pro-apoptotic activity because it binds to and antagonizes the anti-apoptotic activity of Mcl-1 better than 3F-Bok^WT. To validate this idea, we examined 3F-Bok^Δ™ since the interaction between Bok and Mcl-1 is dependent on the Bok TM domain (Lucendo et al., 2020; Szczesniak et al., 2021b; Sancho and Orzaez, 2021). Indeed, 3F-Bok^Δ™ did not trigger apoptosis (Figure 7C, lane 3), or interact with exogenous Mcl-1 (Figure 7D, lane 4), and was not stabilized by Mcl-1 co-expression (Figures 7C,D). Overall, these data show that the Bok TM domain is required for apoptotic signaling and the interaction with Mcl-1, while R200 and K203 of Bok may tune its ability to network with Mcl-1 and influence apoptosis.