Ubiquitin

RRMECs and retinas were lysed in cold IP lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, pH 7.6) containing protease inhibitors (Sigma). Primary antibodies for proteins of interest for immunoprecipitation (PECAM-1, β-catenin) were incubated with SureBeads magnetic beads (Bio-rad) for 10 minutes at room temperature. Equal amounts of retinal or cell lysates were added to the antibody-beads complex, and incubated for 1 hour at RT. To elute the pulled down protein, 1X Laemmli buffer was added to the beads, heated to 75 °C for 15 minutes, and samples were subjected to SDS-PAGE western blot analysis. Primary antibodies against the proteins being investigated for interaction (PECAM-1, β-catenin, SHP2, Ubiquitin (Enzo-life sciences, Farmingdale, NY)) were added to the membranes for incubation at 4 °C overnight, and HRP-conjugated secondary antibodies were added for one hour at room temperature. Specific bands were imaged using the ChemiDoc XRS gel imaging system, and analyzed using densitometry analysis (ImageJ).

Expression vectors for human UCH_L1, UCH_L3, ATXN3, OTUB1, USP5, USP17, USP22, USP25, USP37, USP48 (Sowa et al., 2009 (link)) and TDP43 (Yang et al., 2010 (link)) were obtained from Addgene. Other vectors were described in (Kokura et al., 2010 (link)). USP2A and USP7 cDNAs were from Jiandong Chen (Moffitt). cDNA of E2 enzymes were generated by PCR and subcloned into pCAG vector. Mutations were generated by overlapping PCR and verified by sequencing. The following antibodies were used: Ubiquitin (Enzo), β-actin (Sigma), histone H3 (Abcam), H3K9me3 (Abcam), Flag (Sigma), Myc (Santa Cruz), HA (Covance), GST (Covance), His (Covance), UBE2E (Boston Biochem). SETDB1 antibody was described in (Wang et al., 2003 (link)).