Ca++ contributes to DCLF/IFN-mediated phosphorylation of STAT-1 at Ser 727. HepG2 cells were treated with VEH (0.1% DMSO), (A) BAPTA/AM (10 μM, 4 h before addition of DCLF/cytokines) or (B) 2-APB (100 μM, simultaneous addition with DCLF/cytokines) and treated with sterile water (Control) or DCLF (250 µM) alone or in combination with TNF and/or IFN. Proteins were collected 18 h after drug treatment. pSTAT-1 (Tyr 701), pSTAT-1 (Ser 727), and α-tubulin levels were detected via western analysis. a, significantly different from corresponding bar in VEH group. b, significantly different from corresponding bar in TNF group. c, significantly different from Control within a cytokine group. d, significantly different from DCLF within a cytokine group. Western analysis of proteins from cells treated with and without BAPTA/AM or 2-APB was performed simultaneously. Data are represented as mean ± SEM of at least 3 experiments. Abbreviations: VEH, vehicle; DCLF, diclofenac; pSTAT-1, phosphorylated signal transducer and activator of transcription-1; Tyr, tyrosine; Ser, serine; BAPTA/AM, acetoxymethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; APB, aminophenoxydiphenyl borate.