Fig. 7

CCL22 induces membrane recruitment of DGKα via the Ca²⁺/PLC-γ1 axis. A Silencing of PLC-γ1 in ESCC cells transfected with the two indicated siRNAs was analyzed by immunoblotting. GAPDH was used as the loading control (upper panel). Control or CCR4-treated PLC-γ1 siRNA-transfected KYSE410 and KYSE510 cells were pretreated with the Ca²⁺ chelator BAPTA-AM (10 μM) for 90 min and then incubated with CCL22 (50 ng/ml). Protein lysates were immunoprecipitated with an anti-DGKα antibody and then subjected to immunoblotting to assess the level of pDGKα Tyr³³⁵. B–D Control or CCR4-treated PLC-γ1 siRNA-transfected KYSE410 and KYSE510 cells were pretreated with the Ca²⁺ chelator BAPTA-AM (10 μM) for 90 min and then incubated with CCL22 (50 ng/ml). B Cell membrane and cytosolic proteins were extracted, and immunoblotting was used to detect the expression of DGKα, α1-ATPase (a membrane marker) and TUBA1A (a cytoplasmic marker). C Immunoprecipitation-immunoblotting was used to evaluate the interaction between DGKα and FAK at the plasma membrane. D FAK activity was assessed by ELISA. E The transfection efficiency of the DGKα Y335F plasmid in the indicated ESCC cells was evaluated. GAPDH was used as the loading control. F–H Vector or DGKα Y335F mutant KYSE410 and KYSE510 cells were treated with CCL22 (50 ng/ml). F Cell membrane and cytosolic proteins were extracted, and immunoblotting was used to detect the expression of DGKα, α1-ATPase (a membrane marker) and TUBA1A (a cytoplasmic marker). G Immunoprecipitation-immunoblotting was used to evaluate the interaction between DGKα and FAK at the plasma membrane. H FAK activity was assessed by ELISA. ***P < 0.001, two-tailed unpaired Student’s t test. Error bars, mean ± SD of five independent experiments