Anti myc

The IP assay was conducted with transgenic plants that overexpressed Myc/FLAG-tagged versions of VPS23A. Total proteins were extracted from 10-day-old transgenic seedlings with native buffer (50 mM Tris-MES [pH 8.0], 0.5 M sucrose, 1 mM MgCl 2 , 10 mM EDTA) supplemented with 5 mM DTT, 1 mM PMSF, protease inhibitor cocktail cOmplete Mini tablets (one per 10 ml) (Roche) and 0.2% NP40. The supernatants were collected after centrifugation at 12 000 g at 4 C three times for 5 min each time. Then, the 26S proteasome inhibitor MG132 was added to the sample extracts of Myc-VPS23A, and they were incubated with anti-Myc/Flag magnetic beads (ChromoTek) or p62-agarose (Enzo Life Sciences) at 4 C for 2 h. After washing three times with PBS buffer (pH 7.4), the bead-bound proteins were eluted in sodium dodecyl sulfate (SDS) sample buffer. The sample was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) for 10 min. For ubiquitination analysis, western blots were performed with anti-Myc (Easy Bio), anti-FLAG (Sigma), anti-Ub (stock from our own laboratory), anti-K48-linkage-specific polyubiquitin chains (anti-K48-Ub, Cell Signaling Technology), and anti-K63-linkagespecific polyubiquitin chains (anti-K63-Ub, Cell Signaling Technology) antibodies. For LC-MS/MS analysis, the gel was stained with Coomassie brilliant blue to obtain the candidate interacting proteins of VPS23A.