Western Blot Protein Extraction and Analysis

Reduced protein extracts (typically 10–20 μg protein unless stated otherwise) or immunoprecipitates (IPs) were separated on 8% SDS–PAGE gels, or 4–12% NuPAGE bis–tris precast gels (Invitrogen) by electrophoresis. Proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were blocked in 5% (w/v) non-fat milk (Marvel) in TBS-T (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.2% Tween-20) and incubated overnight at 4 °C in 5% BSA-TBS-T or 5% milk-TBS-T with the appropriate primary antibodies. For ubiquitin blots, membranes were denatured in 6 M guanidine hydrochloride (6 M guanidine HCl, 20 mM Tris–HCl pH 7.5) after transfer just prior to blocking [36 (link)]. Membranes were then washed in TBS-T and incubated with HRP-conjugated secondary antibodies in 5% milk-TBS-T for 1 h, before a further washing in TBS-T and detection using enhanced chemiluminescence reagent (Thermo Scientific) and exposure on medical X-ray films (Konica Minolta) as described previously [37 –39 (link)].