Opposite effects of HGAL and Grb2 on BCR-induced intracellular signaling. (A) BCR-induced intra- and extracellular Ca²⁺ mobilization of the indicated Raji cells recorded by flow cytometry. Different colors indicate different cell lines used in the experiments, as shown in the figure. (B) Raji cells used in panel A were stimulated with anti-human IgM F(ab′)₂ for 5 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 normalized for actin content. The value 1 was assigned to each protein in Raji mock cells. No phosphorylation is observed in unstimulated cells (not shown). (C) 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 (blue), HGAL (green), Grb2(W193K) (red), and a combination of HGAL and Grb2(W193K) (purple). (D) U2932 cells used in panel C 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 and BLNK normalized for actin content. The value 1 for each protein was assigned to U2932 cells transfected with mock plasmid and stimulated for 2 minutes. Data in panels A-D are representative of 3 independent experiments. AU, arbitrary units.