Immunoblotting of Ubiquitination Pathway Proteins

Purified His₆-MavC was used to raise rabbit specific antibodies using a standard protocol (Pocono Rabbit Farm & Laboratory). The antibodies were affinity purified as describe^{38 (link)}. For immunoblotting, samples resolved by SDS-PAGE were transferred onto 0.2 μm nitrocellulose membranes (Pall Life Sciences cat# 66485). Membranes were blocked with 5% non-fat milk, incubated with the appropriate primary antibodies: anti-UbE2N (Cell signaling, cat# 6999S, Thermo Fisher Scientific, cat# 37–1100), 1:1,000; anti-Flag (Sigma, Cat# F1804), 1: 2000; anti-HA (Santa Cruz, cat# sc-7392 1:1,000, Roche, cat# 11867423001 1:5,000), anti-ICDH^{39 (link)}, 1:10,000, anti-actin (Sigma, cat# A2103), 1:5,000, anti-tubulin (DSHB, E7) 1:10,000, anti-Ub K63 (Millipore, cat# 05–1308), 1:1,000, anti-IκBα (Cell signaling, cat# 9242S), 1:1,000, anti-Ub (Santa Cruz cat#sc-8017), 1:1,000, anti-p-IκBα (Cell Signaling, cat# 9246S), 1:1000, anti-UbE2K (Cell Signaling, cat# 8226S), 1:1000, anti-UbE2S (Cell Signaling, cat# 11878S), 1:1000, anti-UbE2E2 (Abcam, cat# Ab177485), 1:1000, anti-His (Sigma, cat# H1029), 1:10,000, anti-p65 (Cell signaling, cat# 8242S), 1:500. Membranes were then incubated with an appropriate IRDye infrared secondary antibody and scanned using an Odyssey infrared imaging system (Li-Cor’s Biosciences).