Anti ha agarose beads

Twenty-four hours post-transfection with constructs indicated, HEK293T cells were cultured in 0.1% serum containing medium for 24–28 h. Cells were then collected and lysed in 1× Cell Lysis Buffer (Cell Signaling Technology) containing protease inhibitors (Roche, Branchburg, NJ) followed by brief sonication. For pulldown of HA- or FLAG-tagged proteins, cell extracts were incubated with Anti-HA agarose beads (Vector Laboratories, Burlingame, CA) or Anti-FLAG M2 magnetic beads (Sigma-Aldrich) overnight at 4 °C. For pulldown of endogenous NFκB2/p100 protein complex, cell extracts were incubated with protein A/G magnetic beads and anti-NFκB2 antibody (1:50 dilution; Cell Signaling Technology; #4882) or anti-rabbit IgG (Cell Signaling Technology; #7074) overnight at 4 °C. Following a brief centrifuge, the supernatant was saved as depleted product for immunoblotting analysis (named as Immunodepletion Assay). The beads were washed three times with 1× Cell Lysis Buffer, and the bound proteins were eluted by boiling in 3×SDS sample buffer (Cell Signaling Technology) and subjected to immunoblotting assay.