HGAL palmitoylation and myristoylation regulates its interaction with Syk. (A) U2932 cells stably expressing WT or G2A HGAL, C43A/C45A HGAL, and G2A/C43A/C45A HGAL mutants were left unstimulated (−) or stimulated (+) with anti-human IgM F(ab′)2 (a-IgM) for 5 minutes. Whole-cell lysates were prepared, immunoprecipitated with V5-tag antibody, separated by SDS-polyacrylamide gel electrophoresis, and immunoblotted with antibodies to Syk or HGAL. Results are representative of 3 independent experiments. (B) U2932 cells stably transfected with mock vector or HGAL and its mutants were left unstimulated (“resting”) or stimulated for 1 minute with goat F(ab')2 anti-human IgM. Western blot analysis of pSyk (Y352), Syk, pBLNK (Y84), BLNK, and HGAL were performed. Actin was blotted to demonstrate equal loading. Results are representative of 3 independent experiments. (C) Kinetic analysis of calcium mobilization in U2932 cells stably transfected with HGAL or its mutants. Horizontal arrow indicates the whole process was in the presence of 1 mM EGTA; vertical arrow indicates the time points at which goat F(ab')2 anti-human IgM and calcium chloride (CaCl2) were added.
