Anti ptp1b

Proteins (50 μg) were extracted from FFPE tissues and separated as mentioned. Primary antibodies were incubated overnight at 4°C as follows: anti-Ephrin-A3 (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PTP1B (1:200 dilution; Abcam, Cambridge, UK), and anti-VEGF (1:200 dilution; Abcam, Cambridge, UK). Secondary antibodies were used as follows: anti-rabbit for Ephrin-A3, PTP1B, and VEGF (1:2,000 dilution, Cell Signaling Technology, Danvers, MA, USA). Beta-actin was used as a loading control.

The antibodies used in the study: mouse monoclonal anti-phosphotyrosine antibody (catalog 05–321, clone 4G10; Millipore); anti-pY1162/1163-IR-β (catalog 700393, clone 97H9L7; Invitrogen); anti-IR-β (catalog sc-711, clone C711; Santa Cruz Biotechnology Inc.); anti-actin (catalog A2228, clone AC-74, Sigma-Aldrich); anti-p-T308 AKT (catalog 13038, clone D25E6; Cell Signaling Tech), anti-AKT (catalog 4691, clone C67E7; Cell Signaling Tech), anti-JAK2 (catalog 3230, clone D2E12; Cell Signaling Tech); anti-catalase (catalog ab16731; abcam), anti-HA (catalog 11583816001, clone 12CA5; Roche), and anti-PTP1B (EP1841Y; abcam). Antibodies were used at dilutions recommended by the manufacturers.

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).