Fig. 7. CLC and sCNTFR co-expression in Cos-7 cells is required for functional responses. (A) STAT3 tyrosine phosphorylation in the HepG2 cell line. HepG2 cells were incubated for 10 min in the presence of LIF (50 ng/ml), purified recombinant sCNTFR (indicated with an asterisk) (1 µg/ml), CNTF (50 ng/ml), CNTF plus exogenous purified recombinant sCNTFR, purified CLC (50 ng/ml), purified CLC plus exogenous purified recombinant CNTFR, or co-synthesized CLC–sCNTFR (50 ng/ml) purified from stably transfected cells. After cell lysis in 1% NP-40, STAT3 proteins were immuno precipitated with the 4G10 anti-phosphotyrosine antibody, analysed by SDS–PAGE, and western blotted with an anti-STAT3 polyclonal antibody. (B) Proliferative response of Ba/F3 cells transfected with gp130 and LIFR to CLC plus exogenous purified recombinant sCNTFR. Cells were cultured in triplicate using 3-fold dilutions of sCNTFR and a fixed amount (20 ng/ml) of CNTF, CLC or IL-2. After a 72 h culture period, a [³H]Tdr pulse was performed and the amount of incorporated radioactivity was determined using a β-counter. Vertical bars indicate the SEM.