Immunoblotting
Purified B cells from WT and CCR2 KO mice were activated at 37 °C with
sAg. At the 5-, 10-, and 30-min activation time-points, 1 ml of PBS was
added to terminate the activation. Cell lysates were obtained using a
mixed RIPA buffer (P0013B, Beyotime, Shanghai, China), protease
inhibitor cocktail (G2006, Servicebio, Wuhan, China), NaF (1 M, G2007-1,
Servicebio), and Na3VO3 (100 Mm, G2007-1, Servicebio). Lysates were run
through SDS-PAGE and western blotting. In addition to the Abs mentioned
in the CFm assay, some other Abs were used. From Cell Signaling
Technology: Btk (8547S), pAkt (4060L), Akt (9272S), SHIP (2728S),
pFoxo-1 (9461S), Foxo-1 (2880S), pS6 (4856S), S6 (2217S), pPI3K (4228S),
PI3K (4292S), pMst1 (3681S), Mst1 (PA5-22015), pmTOR (5536S), mTOR
(2983S), pEzrin (3726S), Stat1 (14994S), P65 (4764S), pIKKB (2697S), and
IKKB (8943S). From Abcam: Stat5 (ab194898). From Santa Cruz
Biotechnology (Santa Cruz, CA, USA): WASP (sc-13139). Loading controls:
anti-mouse β-actin (60008-1-IG-10, Proteintech, Chicago, IL, USA).
Western blotting imaging was performed using the ChemiDoc™XRS + imaging
systems (Bio-Rad, Hercules, CA, USA).