Anti ha 12ca5

Expression vectors for indicated proteins were transiently transfected into human 293 T embryonic kidney cells (ATCC CRL-3216) by the calcium phosphate coprecipitation method^{46 (link)}. Approximately 40 h later, cells were lysed^{47 (link)} and immunoprecipitations performed as previously described^{48 (link)} with either anti-Flag M2 (Sigma-Aldrich F1804) or anti-HA 12CA5 (Santa Cruz Biotechnology sc-57592) mouse monoclonal antibodies. Immunoprecipitates as well as inputs were boiled in Laemmli sample buffer^{49 (link)} and subjected to polyacrylamide gel electrophoresis^{50 (link)}. Separated proteins were transferred to polyvinylidene difluoride membranes^{51 (link)} and challenged with anti-Flag (Sigma-Aldrich F7425) or anti-Myc (Santa Cruz Biotechnology sc-789) rabbit polyclonal antibodies or anti-HA 12CA5 or anti-Myc 9E10 (Sigma-Aldrich M4439) mouse monoclonal antibodies^{52 (link)}. This was followed by incubation with horseradish peroxidase-coupled secondary antibodies^{53 (link)} and signal detection through enhanced chemiluminescence^{54 (link)}.

Anti-HA (12CA5) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) or from Millipore-Sigma (H6908) (Oakville, ON, Canada). The antibody against GFP (11814460001) was purchased from Roche (Oakville, ON, Canada) and used to detect YFP-tagged proteins since the two proteins are identical except for one amino acid and that anti-GFP antibody perfectly recognizes YFP. The antibody against mCherry was purchased from Sigma-Aldrich (AB356482) (Aokville, ON, Canada). Anti-STAU1 was previously described [51 (link)]. Anti-β-Actin (A5441) was obtained from Sigma (Aokville, ON, Canada). All primary antibodies were used at 1:1000 dilution. MG132 (C2211) was purchased from Millipore-Sigma (Aokville, ON, Canada) and used at 20 μM for 8 h. DMSO was purchased from Millipore-Sigma (Aokville, ON, Canada).

Anti-p53 (DO-1) (sc-126, Santa Cruz Biotechnology), anti-Ku70 (A-9) (sc-5309, Santa Cruz Biotechnology), anti-HA (12CA5) (sc: 57592, Santa Cruz Biotechnology), anti-acetyl-H4 (Upstate) antibodies. Filters were reprobed with anti-β-actin mouse monoclonal antibody (Sigma-Aldrich, 63103 USA) or anti-Sp1 mouse monoclonal antibody (Santa Cruz Biotechnology) to normalize respectively cytoplasmic or nuclear protein levels. Anti-H4 (F-9) (sc: 25260, Santa Cruz Biotechnology) mouse monoclonal antibody was used as internal control for acetylated H4. Filters were developed using an enhanced chemiluminescence system (Bio-Rad Clarity Western ECL Blotting Substrates).