IL-6 induces phosphorylation of the C-terminal tyrosine residue of the HGAL protein that mediates increased interaction with myosin protein. (A) Schematic representation of HGAL protein, its deletion mutant missing the C-terminal ITAM (HGAL-ΔC), and substitution mutants in which C-terminal tyrosine residues of the ITAM were substituted with phenylalanine (HGAL-Y128F and HGAL-Y148F). (B) Mutant missing the C-terminal ITAM (HGAL-ΔC) can be expressed in the cellular context. Whole-cell lysates were extracted from SUDHL6, HeLa-HGAL, and HeLa cells transiently transfected with the HGAL-ΔC vector and blotted with the indicated antibodies. (C-E) HeLa cells, transiently transfected with HGAL-ΔC (C), HGAL-Y148F (D), or HGAL-Y128F (E) vectors, were stimulated with IL-6 (10 ng/mL) for up to 60 minutes. Cellular lysates were extracted at the specified time points, immunoprecipitated with anti-HGAL antibody, resolved on SDS-PAGE, and blotted with the indicated antibodies. In panels C-E, representative blots of 3 independent experiments are shown.