Mouse monoclonal anti flag tag antibody

Tris and diethypyrocarbonate were purchased from Amresco (Solon, OH, USA). Mouse anti-Flag-tag monoclonal antibody, anti-Flag M2-agrarose, protease inhibitor cocktail, leupeptin, phenylmethylsulfonyl fluoride (PMSF), Triton X-100, Tween-20, Nonidet P40 (NP-40), hydroxyl urea (HU), ribonuclease A (RNase A), bovine serum albumin (BSA), paraformaldehyde, imidazole, sodium dodecyl sulfate (SDS), guanidine hydrochloride (GdnHCl), polyadenylic acid potassium salts (catalog number: P9403-25MG), DTT, kanamycin, ampicillin and isopropyl-1-thio-β-D-galactopyranoside (IPTG) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-PARN and mouse anti-nucleophosmin (NPM1) antibodies were from Abcam (Cambridge, MA, USA). FITC AffiniPure goat anti-mouse IgG, FITC AffiniPure goat anti-rabbit IgG, mouse anti-HA, rabbit anti-HA, DyLight 594 AffiniPure goat anti-mouse, DyLight 594 AffiniPure goat anti-rabbit, HRP AffiniPure goat anti-mouse and HRP AffiniPure goat anti-rabbit antibodies were from EarthOx (Millbrae, CA, USA). The Annexin V-FITC/PI apoptosis detection Kit was obtained from Bioworld (Louis Park, MN, USA). The transfection reagent VigoFect was from Vigorous (Beijing, China). Hoechst 33342 was from Invitrogen (Carlsbad, CA, USA). All other reagents were local products of analytical grade.

HEK293T cells were seeded in 10-cm cell culture dishes (Thermo Fisher Scientific) at 2 × 10⁶ cells per dish. After culturing for 14 h, 10 µg of each constructed bait plasmid and the constructed prey plasmids were cotransfected into the cells. After 48 h, the cells were washed with cold PBS twice and lysed with 600 μL of IP/lysis buffer (Thermo Fisher Scientific) on ice containing phosphatase inhibitor cocktail (Roche, #4906845001) and protease inhibitor (Roche, #4693132001). Then the cell lysates were centrifuged at 16,000×g at 4 °C for 10 min. The immunoprecipitation experiment was performed with mouse anti-FLAG tag monoclonal antibody (Sigma, #F1804) using a Pierce™ Co-Immunoprecipitation kit (Thermo Fisher Scientific, #26149), and the procedure was performed based on the protocol provided by the kit. Next, empty vectors (p3 × FLAG-CMV and pcDNA3.1+) which served as negative controls, were also cotransfected into HEK293T cells. The elution of the Co-IP was subjected to western blotting and checked with the mouse anti-FLAG tag monoclonal antibody and rabbit anti-MYC tag monoclonal antibody (CST, #2278S).