Mcl 1 d35a5 5453

Samples for SDS-PAGE electrophoresis were prepared in the same way as previously described (Ciołczyk-Wierzbicka et al. 2012 (link)). Antibodies against Phospho-Bcl-2 (Ser70) (SH2) #2827, Phospho-Bcl-2 (Thr56) #2875, Bcl-2 (D55G8) 4223#, Bcl-xl (54H6) 2764#, Mcl-1 (D35A5) 5453# (Cell Signaling Technology), and β-actin (A2228, SIGMA) were used to detect the indicated proteins. Bands were visualized with the use of horseradish peroxidase-coupled secondary anti-mouse or anti-rabbit antibody (Cell Signaling Technology). Immunoreactivity of protein was detected with the use of chemiluminescence, and images were captured with a ChemiDoc MP Imaging System (Bio-Rad Labs). To obtain quantitative results, immunoblots were scanned with the use of SynGene Gene Tools version 4.03.0 (Synoptics Ltd. Beacon House, Nuffield Road Cambridge, CB4 1TF, UK). Densitometry was performed to normalize to β-actin protein level. Presented are representative membranes of at least three independent experiments.

Cells were extracted by direct lysis into SDS‐urea electrophoresis sample buffer, and western blotting was carried out as previously described [52 (link)]. To expose epitopes, membranes to be probed for ubiquitin were boiled in de‐ionised water for 30 min before blocking. The primary antibodies used were p53 (SAPU), Scottish Antibody Production Unit (Carluke, UK); MDM2 (4B2), Moravian‐Biotechnology (Brno, Czech Republic); S5A (14899‐1‐AP), Proteintech Group (Rosemont, IL, USA); ADRM1 (ab56852), RAD23A (EPR4817, ab108591), RAD23B (ab3835), HA tag (HA.C5, ab18181), NOXA (114C307, ab13654) and beta‐actin (ab8226), Abcam (Cambridge, UK); Ubiquitin (P4D1‐A11, 05‐944), p21 (EA10, OP64/Ab‐1) and GFP (mixture of clones 7.1 and 13.1, 11814460001), Merck Group; c‐MYC (9E10), prepared in house and MCL‐1 (D35A5, #5453), Cell Signaling Technology (Danvers, MA, USA). Suitable exposures of western blots were quantified by densitometry using Quantity One, Bio‐Rad (Hercules, CA, USA). SeeBlue Plus2 protein standards were obtained from Thermo Fisher Scientific.