Fig. 6

CCL22 induces the assembly of the CCR4/DGKα/FAK complex in ESCC cells. A Immunoprecipitation-immunoblotting showed that CCR4 interacted with only the FAK C-terminal and not the FAK N-terminal, PI3K catalytic subunit p110α, PI3K regulatory subunit p85, or AKT in KYSE410 and KYSE510 cells with the indicated treatment. B Silencing of DGKα in ESCC cells transduced with the two indicated short hairpin (sh) RNAs was analyzed by immunoblotting. GAPDH was used as the loading control. C The indicated control shRNA- and DGKα shRNA-transduced ESCC cells were treated with PBS or CCL22 (50 ng/ml). The activity of FAK and AKT was assayed by a FAK Tyr³⁹⁷ (left panel) or an AKT Ser⁴⁷³ (right panel) activation quantitative ELISA. D–F Control and CCR4-depleted ESCC cells were treated with or without CCL22 (50 ng/ml). D Silencing of CCR4 in ESCC cells transfected with the two indicated small interfering (si) RNAs was analyzed by immunoblotting. GAPDH was used as the loading control. E The Intracellular Ca²⁺ concentration was evaluated using a calcium detection assay kit. F Protein lysates were immunoprecipitated with an anti-PLC-γ1 or anti-DGKα antibody and then subjected to immunoblotting to evaluate the protein levels of pPLC-γ1 Tyr⁷⁸³ and pDGKα Tyr³³⁵. G KYSE410 and KYSE510 cells were pretreated with the Ca²⁺ chelator BAPTA-AM (10 μM) for 90 minutes and then incubated with CCL22 (50 ng/ml). Lysates were collected to evaluate the expression of pPLC-γ1 Tyr⁷⁸³ and PLC-γ1 using immunoblotting. H Immunoprecipitation-immunoblotting showed that PLC-γ1 interacted with DGKα but not FAK, CCR4, or AKT in KYSE410 or KYSE510 cells treated with CCL22 (50 ng/ml)