PTP1BΔ6:PTP1BWT dimer formation attenuates PTP1B activity. (A) Protein complex formation of HA-PTP1BWT with FLAG-PTP1BWT, FLAG-PTP1BΔ6, or FLAG-PTP1BC215S. WCE from HEK293 cells ectopically expressing HA-PTP1BWT either alone or in combination with FLAG-PTP1BWT, FLAG-PTP1BΔ6, or FLAG-PTP1BC215S were subjected to an immunoprecipitation analysis using an anti-HA antibody. Coprecipitated FLAG-PTP1B variants were visualized by an anti-FLAG immunoblot analysis. (B) Phosphatase assay with EXPRESS-tagged PTP1BWT alone or together with HA-PTP1BC215S, or increasing amounts of HA-PTP1BWT, or HA-PTP1BΔ6 ectopically expressed in HEK293 cells, as indicated. pEXPRESS (EV) serves as control. Mean values and SEM are depicted. Significances are calculated in comparison with the empty vector values. *P ≤ .05; **P ≤ .01; ***P ≤ .001. (C) Luciferase assay to determine the STAT6 activity in HEK293-ST6 cells with and without (control) stimulation with IL-4. The cells were transiently transfected with 10, 50, or 100 ng FLAG-PTP1BWT, FLAG-PTP1BC215S, or FLAG-PTP1BΔ6. The resulting WCE were additionally tested for the expression of exogenous PTP1B (anti-FLAG IB) or overall PTP1B (anti-PTP1B IB). The full black circle indicates the position of the endogenous PTP1B, the open circle indicates the position of the exogenous PTP1BΔ6 (lower-middle, last 3 lanes). The membrane was reprobed, using an anti-β-actin antibody to ensure equal protein loading. Mean values and SEM are depicted. Significances are calculated in comparison with the empty vector values. *P ≤ .05; **P ≤ .01; ***P ≤ .001. (D) Luciferase assay for STAT6 activity with either unstimulated cells (control) or cells stimulated with either 1 ng/ml or 5 ng/ml IL-4. HEK293-ST6 cells were transiently transfected with 100 ng HA-PTP1BWT, HA-PTP1BΔ6, or HA-PTP1BC215S. pcDNA3.1 (EV) serves as control. Mean values and SEM are depicted. Significances are calculated in comparison with the empty vector values. *P ≤ .05; **P ≤ .01; ***P ≤ .001.
