BOK Antibody - #DF3829

Fig. 3 SARS-CoV-2 M protein inhibited BOK ubiquitination and promoted its translocation and interacted with BOK via endodomain. A, B H292 cells were not transfected (control) or transfected with an empty vector (pcDNA6B) or M-Flag plasmid for 48 h. The protein levels of Bcl-2, BID, BAX, BAK and BOK (A) or M-Flag, Precursor caspase-3 (Pro-CASP 3) and cleaved CASP 3 were analyzed by WB. C After H292 cells were transfected with M-Flag for 48 h, the mitochondrial and cytosolic fractions were isolated and analyzed by WB. D mRNA levels of Bok in H292 cells transfection with M-Flag for 48 h were examined by qPCR. E Cycloheximide (CHX) chase to check the turnover of endogenous BOK, H292 cells either transfected with vector or M-Flag expressing plasmid, were treated with 30 μg/ml CHX and harvested at the indicated time periods for WB analysis (upper panel), and quantification of BOK level. β-actin use as internal control (lower panel). F Lysates from H292 non-transfected (control) or transfected with an empty vector (pcDNA6B) or M-Flag plasmid followed by treatment with 10 μM MG132 for 5 h before harvest and were immunoprecipitated. Ub Ubiquitination. G HEK293T cells were cotransfected with plasmids encoding HA-tagged ubiquitin, Myc-tagged BOK, together with either an empty vector (pcDNA3.1(+)) or plasmid encoding Flag-tagged M protein for 40 h. Cells were treated with 10 μM MG132 for 5 h and lysed. Then the proteins mixture was immunoprecipitated with anti-Myc antibody followed by WB. H Immunoprecipitation using anti-Flag or anti-HA antibodies from lysates of 293 T cells transfected with HA-tagged BOK (24 kDa) alone, or with an empty vector (pcDNA6B), SARS-CoV-2 Flag-tagged N (51 kDa) and M (23/24 kDa). I Exogenous protein interaction was further confirmed in 293 T lysates immunoprecipitated with anti-HA and analyzed by WB with the indicated antibodies. J, K 293 T cells were co-transfected with pcDNA3.1(+) carrying HA-tagged BOK, with a pcDNA6B vector containing Flag-tagged M (full lengh), Flag-tagged truncated variants of M proteins (M-▵transmembrane (TM)1-Flag (22 kDa), M-▵TM1-2-Flag (18/20 kDa), M-▵TM1-3-Flag (14 kDa), M-▵endodomain (ED)-Flag (22 kDa)). Blots of cell lysates (input) or anti-Flag (J) and anti-HA (K) immunoprecipitates were analyzed by Western blotting for HA and Flag. L Confocal microscopy of 293 T cells co-transfected with HA-tagged BOK and Flag-tagged M (above lane) or Flag-tagged M-▵ED (below lane). White arrows represent the colocalization. Scale bars, 10 μm. Data present with mean ± SD for at least three independent experiments. ns non-significant, **p < 0.01. Data were analyzed with one-way ANOVA with LSD’ t test.