HGAL colocalizes with Grb2 at cSMACs and facilitates antigen aggregation in a planar lipid bilayer model. (A-B) Upon antigen stimulation of wild-type HGAL expressing U2932 (A) and Bjab (B) cells, BCR microclusters formed and eventually accumulated in cSMACs at 30 minutes. HGAL colocalizes with the BCR in cSMACs; Grb2 redistributes to the membrane area and colocalizes with HGAL in cSMACs (top panels). In HGAL (FEN) mutant–expressing cells, HGAL mutant exhibits only pSMAC distribution and lost cSMAC colocalization with Grb2 and results in less defined cSMACs with decreased BCR accumulation (lower panels). (C-D) BCR microcluster/cSMAC formation as a function of time in U2932 (C) and Bjab (D) cells. Error bars denote ±1 standard error of the mean. Two-way analysis of variance was performed to determine if the kinetic responses of U2932 and Bjab cells transfected with HGAL and HGAL (FEN) are different. P = .0001 for U2932 and P = .0001 for Bjab cell lines. (E) Distribution of HGAL, HGAL(FEN), Grb2, and BCR in Raji HGAL KO cells reconstituted with wild-type HGAL-GFP and HGAL(FEN) GFP, Riaji Grb2 KO, and Raji HGAL and Grb2 KO cells 30 minutes after stimulation. Each image shown in panels A-B,E is 20 microns wide.