Protein Aggregation and Ubiquitination Analysis

All cell culture and standard reagents were purchased from Sigma. The region of bovine opsin used as an N-glycosylation reporter and epitope tag was as described previously (Johnson et al., 2012 (link)). Anti-TRC40 serum was a gift from Bernhard Dobberstein (ZMBH, Heidelberg, Germany). Commercially available antibodies against the following targets were purchased from the indicated suppliers: BAG6, BAP31, GFP, tubulin and V5 (Abcam), FLAG M2 and calnexin (Sigma), HSP70 (Stressgen), ubiquitin FK2, p23, 20S proteasome (Enzo Life Sciences) and LAMP1 (DHSB, University of Iowa). The antibody against GRASP65 was a gift from Martin Lowe (University of Manchester, UK). ProteoStat® reagent for the detection of protein aggregates was from Enzo. Bortezomib was from Selleck Chemicals, leupeptin and pepstatin A were from BIOMOL. RFP in pcDNA3.1⁺ was a gift from Viki Allan (Manchester, UK). siRNA duplexes for knockdowns were from Qiagen, and they targeted sequences that were identified previously (Winnefeld et al., 2006 (link)): SGTA target sequence, 5′-TTTGAAGCTGCCGTGCATT-3′; BAG6 target sequence, 5′-CAGCTCCGGTCTGATATACAA-3′.