Rabbit anti ubiquitin

Cells were lysed in whole cell lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% SDS) containing 1X protease and phosphatase inhibitor (Pierce, 78440). Lysates were cleared by centrifugation (12,000g for 10 minutes). Protein quantification was performed using the Pierce BCA protein assay kit according to the manufacturer’s instruction (Thermo Fisher Scientific, 23227). Equal quantities of proteins were mixed with loading dye, and incubated at 95°C for 5 minutes before they were separated on 4–12% Bis–Tris gels (Invitrogen) and transferred onto a PVDF membrane at constant 350 mA at 4°C for 1 hour. Membranes were blocked by incubation in 5% milk for 1 hour at room temperature under agitation and then incubated with the following primary antibodies: mouse anti-GAPDH (Santa Cruz) with a 1:5000 dilution in 1%milk overnight, or rabbit anti-mCherry (Abcam, ab167453) with a 1:1000 dilution in 1%milk overnight, or rabbit anti-ubiquitin (Abcam, ab19247) with a 1:1000 dilution in 1%milk overnight. After incubation with the appropriate secondary antibodies conjugated to HRP and extensive washing, blots were imaged on a ChemiDoc XRS+ system (Bio-Rad).