PTP1B-D181A colocalizes and traps pSTAT6 in the cytoplasm. Control vector-transfected HeLa cells (A) or cells transiently expressing PTP1B D181A mutant (B) were stimulated with IL-4 (100 U/mL) for 15 minutes, fixed in 4% formaldehyde, and stained with DAPI and antibodies to pSTAT6 and PTP1B as described in “Methods” in “Microscopy.” Cells were visualized by Zeiss LSM510 confocal microscope with a plan-apochromat 40×/1.3 NA oil-immersion lens, 364, 488, and 555 nm laser lines, for DAPI, Alexa Fluor 488 detection of anti-STAT6, and Alexa Fluor 555 detection of anti-PTP1B, respectively. Images were acquired using 1024 × 1024 pixel scan size, 12-bit digitization, and 4-frame averaging. The measurements were performed as described in “Methods” in “Microscopy.” (C) Four-fold magnification of individual cells from experimental conditions shown in panels A and B. (A-C) Slides were viewed with a Zeiss LSM 510/UV confocal Axiovert 200M microscope (Zeiss, Thornwood, NY) 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) Pixel intensity corresponding to pSTAT6 was quantified as described in “Methods” in “Microscopy” in 175 control HeLa cells and 175 HeLa cells expressing the PTP1B D181A mutant. The results shown are mean plus or minus SE; P < .001. The results in panels A, B, and D are representative of 3 independent experiments.