Control mouse igg

HUVECs underwent immunoprecipitation (IP) using mouse control IgG (Santa Cruz, USA, sc-2025). For IP, antibodies were coupled to Dynabeads^TM (Invitrogen). 10 million cells were used for IP and separated for control and Rbm38 pulldown group.

To test the 9F mAb in immunoprecipitation (IP) assays, the input sample consisted of adult mouse brain total protein extract (1mg) and was pre-cleared by incubation (1h at 4 °C on a rotating wheel) with 2μg of mouse control IgG (Santa Cruz Biotechnology, sc-2025). The solution was then incubated with 50µl of slurry Protein A/G plus-agarose beads (Santa Cruz Biotechnology, sc-2003) for 1h at 4 °C on a rotating wheel and subsequently centrifuged at 4000xg for 5min at 4 °C. The resulting supernatant was incubated with 10μg of the anti-aldolase C 9F mAb for 1h on a rotating wheel at 4 °C. Then, 50µl of fresh Protein A/G plus-agarose beads were added to the test tube and incubated for 2h at 4 °C. After extensive washing of the pellet beads, the resulting immunoprecipitated complex was eluted incubating the resin at 99 °C for 5min in 40µl of 2X protein sample buffer. Immunoprecipitation lysis buffer was constituted by 10% glycerol, 50mM TRIS-HCl pH 8, 150mM NaCl, 0.1–1% NP40, 1X Complete Roche Protease inhibitors, 0.5mM PMSF. For western blot analysis 1/10 of the total immunoprecipitated fraction was assayed. The 9F mAb was used also in the immunoblot analysis of the immunoprecipitation result.