Anti k48 linked ubiquitin

Cell lysis and immunoblotting was performed as previously described [14 (link)]. Briefly, cells were lysed in PBS supplemented with 0.25% triton X and cOmplete, Mini Protease Inhibitor Cocktail (Roche). Lysates were then sonicated, and then centrifuged at 500×g to remove debris. An aliquot of sample containing total cellular protein was frozen at −80°C. Total cellular proteins, soluble, and membrane proteins were loaded on SDS–PAGE gels (Invitrogen) and processed for immunoblotting using the following antibodies: anti-E-Cadherin (Life Technologies, 13-5700), anti-hemagglutinin, anti-ubiquitin (LifeSensors, VU101), anti-K48-linked ubiquitin (Cell Signaling, 8081S), anti-K63-linked ubiquitin (Cell Signaling, 5621S), anti-CD172a (Millipore, 566310), anti-IFNGR1(Santa Cruz, sc-28363), anti-GSK3β (Cell Signaling, D613M), anti-V5 (Cell Signaling, 13202S), anti-GAPDH (Sigma), anti-STAT-1 (Cell Signaling, 9172P), anti-pSTAT-1 (Cell Signaling, 7649P), anti-EGFR (Cell Signaling, 4267S), and anti-β-actin mouse monoclonal antibody (Sigma). Immunoblots were exposed to Supersignal West Femto chemiluminescent substrate (Thermo Scientific).