M2 anti flag mab agarose beads

For purifying Flag-tagged CCN2 fusion proteins, HEK-293 T cells with Flag-tagged CCN2 stable overexpression were harvested in RIPA lysis buffer supplemented with complete protease inhibitor and phosphatase inhibitor (Roche Applied Science). The lysate was cleared by centrifugation at 12000 g before being loaded to M2 anti-Flag mAb agarose beads (Sigma, St. Louis, MO, USA) pre-equilibrated in RIPA buffer overnight at 4 °C. The beads were washed with RIPA buffer five times and 30ul RIPA buffer was added to cover the beads. The RIPA buffer containing beads and Flag-tagged CCN2 fusion proteins were stored at 4 °C for the next experiment. For the extraction of membrane proteins, MHCC97H cells were grown to 75% confluence. The membrane proteins were extracted using a ProteoExtract Native Membrane Protein Extraction Kit (M-PEK Kit; Calbiochem, La Jolla, CA, USA) according to manufacturer’s instruction. For the binding assay, Flag-tagged CCN2 bind to the beads was incubated with membrane proteins with or without LMWH (2 μg/ml, Santa Cruz Biotechnology) overnight at 4 °C in RIPA buffer. Then, the beads were washed with RIPA buffer five times and bound proteins eluted using Flag peptide (Sigma). The washed protein was boiled in loading buffer, resolved on SDS-PAGE. Subsequent immunoblots were probed with the appropriate antibody and detected by ECL.

MHCC-97L cells with stable overexpression of OPN or empty control transfected with FLAG-tagged vimentin. 24 hours after transfection, cells were incubated with 40 μM MG-132 for 36 hours, then washed twice with PBS. Cell were extracted in 2 ml RIPA lysis buffer supplemented with complete protease inhibitor (Roche Applied Science). Next, 20 μl M2 anti-Flag mAb agarose beads (Sigma, St. Louis, MO) was used to immunoprecipitate FLAG-vimentin protein in the cell extract at 4°C for 3 hours. The beads were washed with RIPA buffer five times and bound proteins eluted using Flag peptide (Sigma). The washed protein was boiled in loading buffer, resolved on SDS-PAGE for detection of vimentin and ubiquitin by Western blot.

Cells were washed twice with ice-cold PBS and then lysed with RIPA lysis buffer supplemented with complete protease inhibitor (Roche Applied Science). The lysate was cleared by centrifugation at 12000 g before being loaded to M2 anti-Flag mAb agarose beads (Sigma, St. Louis, MO) pre-equilibrated in RIPA buffer overnight at 4°C. The beads were washed with RIPA buffer five times and bound proteins eluted using Flag peptide (Sigma). The washed protein was boiled in loading buffer, resolved on SDS-PAGE. Subsequent immunoblots were probed with the appropriate antibody and detected by ECL.