HGAL (FEN) mutant not interacting with the Grb2 protein exhibits enhanced effects on BCR signaling compared with wild-type HGAL. (A) BCR-induced intra- and extracellular Ca²⁺ mobilization of the indicated U2932 cells recorded by flow cytometry. Lines represent U2932 cells transfected with the following plasmids: mock (orange), HGAL (blue), and HGAL (FEN) (red). (B) U2932 cells used in panel A were stimulated with anti-human IgM F(ab′)₂ for 2 and 10 minutes. Whole-cell lysates were prepared, separated by SDS-PAGE, and immunoblotted with the indicated antibodies. Actin was blotted to demonstrate equal loading. Densitometry was measured for phosphorylated Syk, BLNK, BTK, PLCγ2, pJNK, pP38, and pERK normalized for actin content. The value 1 was assigned for each protein in U2932 cells transfected with mock plasmid and stimulated for 2 minutes. Data in panels A-B are representative of 3 independent experiments. (C) U2932 cells used in panel A were cotransfected with both pNF-κB-Luc reporter and pRL-TK plasmids for 24 hours and stimulated with anti-human IgM F(ab′)2 for 30 minutes. Whole-cell lysates were prepared and used for determination of luciferase activity. Numbers refer to relative luciferase activities (Luc/Rlu) representing means + standard deviation of the mean of 3 independent experiments, each performed in triplicate. Asterisks indicate statistically significant differences (*P = .02; **P < .0005).