Fig. 6. IL-6 protects PrLZ from degradation through activating ERK1/2.
a Immunoblot (IB) analysis of WCL derived from C4-2 and 22Rv1 cells treated with different concentration of IL-6 (10, 25, 50 and 100 ng/ml) for 48 h. b IB analysis of WCL derived from C4-2 cells treated with IL-6 (50 ng/ml, 48 h) or/and transfection with HA-SPOP. c IB analysis of WCL derived from C4-2 cells treated with IL-6 (50 ng/ml, 48 h) or/and SCH772984 (3 μM). d IB analysis of WCL and anti-Flag immunoprecipitates (IPs) derived from 293 T cells transfected with HA-SPOP and Flag-PrLZ. Cells were treated with IL-6 (50 ng/ml, 48 h) or/and SCH772984 (3 μM). 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. e IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, HA-SPOP and His-Ub. Cells were treated with IL-6 (50 ng/ml) or/and SCH772984 (3 μM). Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. f IB analysis of WCL derived from C4-2 cells treated with IL-6 (50 ng/ml) or/and SCH772984 (3 μM). Where indicated, 100 μg/ml CHX was added for the indicated time period before harvesting. g The PrLZ protein abundance in (f) was quantified by ImageJ and plotted as indicated. PrLZ bands were normalized to vinculin. h IB analysis of WCL derived from C4-2 cells transfected with Flag-PrLZ WT and Flag-PrLZ S40A. Cells were treated with IL-6 (50 ng/ml) or/and SCH772984 (3 μM). i, j Colony formation assays and quantitative analysis of C4-2 cells transfected with Flag-PrLZ WT or Flag-PrLZ S40A in the presence or absence of IL-6 (50 ng/ml) or/and SCH772984 (3 μM) treatment. **P < 0.05. k–m C4-2 cells transfected with PrLZ WT or PrLZ S40A were subcutaneously injected into nude mice, which were treated with IL-6 (100 ng per mouse) or/and SCH772984 (SCH, 50 mg/kg, daily). Statistical analysis of the tumor volumes which were measured every three days and plotted individually (k). Subcutaneous xenograft tumors formed from different groups in C4-2 cells were dissected (l). Statistical analysis of the weights of the dissected xenografts tumors (m). n = 6 mice per experimental group, the results indicated the mean ± S.D. *P < 0.05.