Anti tcptp

Total protein extracts were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were then blocked with 5% nonfat milk in Tris-buffered saline with Tween-20 for 2 h at room temperature and incubated with anti-PTP1B (Abcam, United States), anti-TCPTP, and anti-GAPDH (Cell Signaling Technology, United States) at 4°C overnight, rinsed three times with TBST, and incubated with respective secondary antibodies for 2 h at room temperature. Protein bands were visualized with Thermo Fisher Scientific SuperSignal West Femto Maximum Sensitivity Substrate (Rockford, United States) and captured using an Image Quant LAS4000 imaging system (Shanghai, China).

Total cell lysates from both keratinocytes and the epidermis were prepared with RIPA buffer (Thermo Fisher Scientific) containing 1% Triton X-100, protease inhibitor cocktail (Sigma-Aldrich), and phosphatase inhibitor cocktail I and II (Sigma-Aldrich). Equal amounts of total protein were resolved using SDS-PAGE and transferred to the PVDF membrane (GE Healthcare). The membrane was incubated overnight at 4 °C with a primary antibody, followed by incubation with a secondary antibody conjugated to horseradish peroxidase. Enhanced chemiluminescence detection reagents (Pierce) were used to detect immunoreactive protein. The following antibodies were used: anti-phospho-Flk-1 (Tyr949) (#2471); anti-phospho-Flk-1 (Tyr994) (#2474); anti-phospho-Flk-1 (Tyr1173) (#2478); anti-Flk-1 (# 9698); anti-phospho-STAT3 (#9145); anti-STAT3 (#9139); anti-phospho-ERK (#4370); anti-ERK (#4695); anti-phospho-JNK (#4668); anti-JNK (#9258); anti-Bcl-xL (#2762); anti-Bax (#2772); anti-TC-PTP (#58935); anti-cleaved caspase 3 (#9664); and anti-PARP (#9542) from Cell Signaling Technology; anti-phospho-Flk-1 (Tyr1052/1057) (#44-1047G); anti-phospho-Flk-1 (Tyr1212) (#44-1052) from Invitrogen; anti-phospho-Flk-1 (Tyr779) (#VP2921) from ECM Biosciences; anti-β-actin (sc-47778) from Santa Cruz Biotechnology; anti-FLAG^® M2 (#F3165) from Sigma-Aldrich; and anti-CD44 (#NBP1-47386) from Novus Biologicals.