Ab31392

The isolation of total protein from UWB1.289 cells was performed using RIPA buffer (Invitrogen). Protein concentrations were measured by performing BCA assay (Invitrogen). Protein samples were denatured in boiling water for 10 min, followed by separation of proteins using SDS-PAGE gel (8%). Proteins were transferred to PVDF membranes, followed by blocking in PBS containing 5% non-fat milk for 2 h at 25 °C. The blocked membranes were first incubated with rabbit primary antibodies of PTEN (ab31392,Abcam) and GAPDH (ab9485, Abcam) at 4 °C for 20 h, followed by incubation with secondary antibody of lgG-HRP (ab6721, Abcam) for 2 h at 25 °C. After that, signals were developped using ECL™ Select Western Blotting Detection Reagent (Sigma-Aldrich). Image J v1.48 software was used to normalize signals.

After transfection and HR treatments, AC16 cells were collected and lysed in 1 × ice-cold RIPA lysis buffer (#P0013B, Beyotime, Shanghai, China). The protein was quantified with the BCA kit (#T9300A, Takara, Dalian, China). A total of 35 μg proteins was separated by electrophoresis on 10% SDS-polyacrylamide gel (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 1 × Tris-buffered saline with Tween (TBST) containing 5% nonfat milk for 1 h at room temperature and incubated with specific primary antibodies from Abcam (anti-Bcl2, #ab59348, 1:1000; anti-Bax, #ab53154, 1:1000; anti-cleaved caspase 3, #ab2302, 1:1000; anti-PTEN, #ab31392, 1:1000; anti-PI3K, #ab70912, 1:1000; anti-p-PI3K, #ab182651, 1:1000; anti-AKT, #ab8805, 1:1000; anti-p-AKT, #ab8933, 1:1000; anti-β-actin, #ab8227, 1:1000) at 4 ℃ overnight. After wash, the membranes were then incubated with the corresponding secondary antibodies for 2 h at room temperature. Finally, the blot was incubated with ECL plus reagent (#32109, Pierce, Rockford, IL, USA) and visualized using charged-coupled device LAS 4000 (Fujifilm, Valhalla, NY, USA) and photographed. All antibodies were purchased from Abcam (MA, USA) and diluted as suggested by the manufacturer.

Western blot analysis was performed as previously described [58 ]. Briefly, cells were lysed in cold lysis buffer, proteins (20–30 μg) were resolved on SDS-PAGE, transferred onto PVDF membranes, and probed with antibodies for EYA2 (ab95875, Abcam), PTEN (ab31392, Abcam), and GAPDH (sc-32233, Santa Cruz Biotechnology) at 4°C overnight. Detection was performed with the SuperSignal West Femto Maximum Sensitivity Substrate Trial Kit (Pierce, Rockford, IL, USA). The band images were digitally captured and quantified with a FluorChem FC2 imaging system (Alpha Innotech, San Leandro, CA, USA). Protein levels were normalized to GAPDH and quantified with respect to vector control group.

Whole-cell lysates were prepared from human NSCLC cell lines after harvesting and centrifuging. Protein concentrations were quantified using BCA kit (Beyotime Bio, Shanghai, China). The proteins were denatured, electrophoresed (SDS-PAGE gel Kit, Beyotime Bio, Shanghai, China), and then transferred to PVDF membranes (Millipore Co., MA, USA). Subsequently, the PVDF membranes were blocked with non-fat milk powder, incubated with primary and secondary (HRP-labeled goat anti-rabbit IgG) antibodies, and visualized (Bio-Rad Image Lab Software).
Western blotting was performed using the primary antibodies, anti-SH2B1 (1:1000, ab196575, Abcam), anti-Akt (1:600, ab8805, Abcam), anti-Akt (phospho S473) (1:300, ab8932, Abcam), anti-mTOR (1:1500, ab2732, Abcam), anti-mTOR (phosphor S2448) (1:1000, ab109268, Abcam), anti-PTEN (1:600, ab31392, Abcam), anti-C-Myc (1:1000, ab32072, Abcam), antibodies (Abcam, Cambridge, MA, USA), and anti-GAPDH antiboty (1:3000. D110016, Sangon Biotech, Shanghai, China). Following the initial western blotting assay, the membranes were stripped and the band intensities were relative to GAPDH. The quantification of protein bands was performed using Image J software.

Paraffin-embedded sections of tumor tissues were stained for PTPN14 and PTEN expression. Immunohistochemistry (IHC) and the scoring system for PTPN14 (1:200, SAB2700311, Sigma-Aldrich, USA) and PTEN (1:100, ab31392, Abcam, UK) were performed as we previously described [50 (link)].