STAT6 is the physiologic substrate of PTP1B. (A) HEK293 cells were transfected with Myc-tagged STAT6 and 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 anti-MYC antibody. (B) HeLa cells were transfected with either SMART pool 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 120 minutes. Cellular lysates were extracted at indicated time points and blotted for pSTAT6, STAT6, PTP1B, and actin. Mean relative densitometry of pSTAT6/STAT6 ratio from 3 independent experiments is depicted. The value in specimen siRNA-PTP1B at time point 0 was arbitrarily defined as 1. (C) HeLa cells were transfected with either SMART pool siRNA for PTP1B or scrambled control (200 pmol). At 48 hours after transfection, the cells were stimulated with IL-4 (100 U/mL) for 15 minutes and fixed in 4% formaldehyde and stained with 7AAD (nuclear staining) and antibodies to pSTAT6 and PTP1B. Slides were viewed with a Zeiss LSM510/UV confocal Axiovert 200M microscope (Zeiss, Thornwood, NJ) using a Zeiss plan-apochrome oil-immersion lens at 40×/1.3 NA and ProLong Gold antifade reagent (Invitrogen, Eugene, OR) as mounting medium. Images were acquired using a Zeiss LSM510/UV confocal scanner, and were processed with Zeiss LSM510 AIM 3.2 SP2 confocal microscope software and Adobe Photoshop version 7.0 (Adobe, San Jose, CA). (D) IL-4 induces enhanced STAT6 phosphorylation in PTP1B-deficient cells. Mouse embryo fibroblasts from PTP1B-deficient (^−/−) and wild-type (^+/+) mice were serum starved for 4 hours and then stimulated with 50 ng/mL IL-4 for the indicated times. Activation of STAT6 and AKT was assessed with phosphotyrosine-specific STAT6 and phosphoserine-specific AKT antibodies. Equal loading was confirmed by immunoblotting with an actin antibody. Representative results of 3 independent experiments are shown.