Dephosphorylation of pSTAT6 by PTP1B is independent of PTP1B effects on JAK1. (A) HEK293 cells were transfected with STAT6 plasmid and either PTP1B plasmid or empty vector. Forty-eight 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-JAK1 or JAK2 antibodies followed by anti- pTyr or anti-JAK1 or JAK2 Western immunoblotting. Corresponding cellular lysates were also immunoblotted with STAT6 and pSTAT6-specific antibodies. (B) HeLa cells were either left unstimulated or were stimulated with IL-4 (100 U/mL) for 30 minutes. Cellular lysates were extracted and subjected to immunoprecipitation with anti- JAK1 and PTP1B followed by anti-PTP1B and anti-JAK1 immunoblotting. (C) HeLa cells were transfected with either SMART pool of siRNA for PTP1B or scrambled control (200 pmol). At 72 hours after transfection, the cells were stimulated with IL-4 (100 U/mL) for 15 to 45 minutes with or without the addition of staurosporine starting 15 minutes after IL-4 stimulation. Cellular proteins were immunoblotted for pSTAT6, STAT6, and actin at the indicated times. Representative results of 3 independent experiments are shown.