PTP1B interacts with the transactivation domain of STAT6. (A) Schematic representation of STAT6 and its mutant missing the C-terminal transactivation domain (STAT6ΔC). (B,C) HEK293 cells were transfected with MYC-fused STAT6 or STAT6 ΔC plasmids and vector control, PTP1B, or PTP1B D181A plasmids. At 48 hours after transfection, cells were stimulated with IL-4 (100 U/mL) for 30 minutes. Cellular lysates were extracted and subjected to immunoprecipitation with (B) anti-MYC or (C) anti-PTP1B antibodies followed by anti-PTP1B and anti-STAT6 Western blotting. (D) HEK293 cells were transfected with PTP-PEST, PTP-PEST-D199A, STAT6 plasmids, and vector control. At 48 hours after transfection, cells were stimulated with IL-4 (100 U/mL) for 30 minutes. Cellular lysates were extracted and subjected to immunoprecipitation with anti–PTP-PEST or anti-STAT6 followed by blotting with anti-STAT6 and anti–PTP-PEST antibodies, respectively. (E) HeLa and OCILY10 cells were stimulated with IL-4 (100 U/mL) for 30 minutes. Cellular lysates were extracted and subjected to immunoprecipitation with anti-STAT6 followed by anti-PTP1B or anti-STAT6 blotting. (F). STAT6 and PTP1B were transiently expressed in the HEK293 cells, as described in “Methods” in “Cell transfections.” At 48 hours after transfection, the cells were left untreated or incubated for 30 minutes with sodium orthovanadate (2 mM). The cells were then stimulated with IL-4 (100 U/mL) for 30 minutes, and cellular lysates were extracted and subjected to immunoprecipitation with anti-MYC followed by blotting with either anti-PTP1B or anti-STAT6 antibodies. Representative results of 3 independent experiments are shown in panels B-F.