Rabbit monoclonal anti syvn1

Cells and tissue samples were lysed with urea lysis buffer or nonidet P‐40, and protein concentrations were quantified. The extracts were incubated with rabbit polyclonal anti‐ubiquitin (Cat#3933, Cell Signaling Technology), rabbit monoclonal anti‐SYVN1 (Cat#14773, Cell Signaling Technology), rabbit polyclonal anti‐actin (Cat#ab8227, Abcam, Cambridge, UK), mouse monoclonal anti‐HSP90 (Cat#sc‐13119, Santa, Dallas, TX, USA), rabbit polyclonal anti‐EEF2K (Cat#3692S, Cell Signaling Technology), mouse monoclonal anti‐Flag (Cat#sc‐51590, Santa), or mouse monoclonal anti‐CD31 (Cat#3528, Cell Signaling Technology) at 4°C for 2 h. Then, protein A/G beads were added to samples and incubated for 2 h. After incubation, protein A/G beads were washed four times with ice‐cold 1 × PBS and heated in boiling water for 5 min with loading buffer. Then, samples were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) on a 4%‐15% gradient gel (Cat#4561085, Bio‐Rad, Hercules, CA, USA). A polyvinylidene fluoride membrane was incubated with the indicated antibodies after transfer and subsequently incubated with corresponding second antibodies and detected using a chemiluminescence kit (Cat#P0018M, Beyotime).