Anti ptpro

Cells were lysed in RIPA buffer (Cell Signaling Technologies). Proteins were fractionated on 4–12% acrylamide gels (Novex, Thermo Fisher Scientific) under denaturing conditions. Antibodies used were anti-RPLP1 (Ab121190; Abcam), anti-RPLP2 (Ab154958; Abcam), anti-mouse β-actin (sc-47778; Santa Cruz Biotechnology), anti-DENV NS3 (GTX124252; GeneTex), anti-FLAG (F7425; Sigma-Aldrich), anti-HA (ab18181; Abcam), anti-TSPAN12 (A05472–1; Boster-Bio); anti-MUC16 (sc-365002; Santa Cruz Biotechnology); anti-XRN1 (sc- 165985; Santa Cruz Biotechnology); anti-SEMA7A (sc-374432; Santa Cruz Biotechnology); anti-MIB1 (sc-393551; Santa Cruz Biotechnology); anti-PTPRO (sc-365354; Santa Cruz Biotechnology); anti-PARD6B (sc-166405; Santa Cruz Biotechnology); anti-eEF2K (sc-390710; Santa Cruz Biotechnology); anti-XRN1(ab70259; Abcam); anti-eEF2 (2332; Cell Signaling Technology); anti-EMC4 (ab184544; Abcam). For quantification, protein from triplicate wells were measured and averaged.

Equal amount of protein extracts were lysed using radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA). Cells were centrifuged in a microcentrifuge at 12,000 × g for 15 min at 4°C to collect the supernatant. Protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 30 µg protein were separated by 10% SDS-PAGE and then transferred onto polyvinyl fluoride membranes (Merck KGaA). The membranes were blocked with 5% fat-free milk in Tris-buffered saline for 1 h, and incubated with anti-PTPRO (1:1,000; catalog no. sc-365354; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin (1:2,000; catalog no. 4970, Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibodies at 4°C overnight. The membranes were washed three times with Tris-buffered saline containing 0.1% Tween and incubated for 2 h at room temperature with a horseradish peroxidase-conjugated goat anti-rabbit (catalog no. 7074; 1:1000; Cell Signaling Technology, Inc.) or anti-mouse secondary antibody (catalog no. sc-516102, 1:2000, Santa Cruz Biotechnology). Proteins were visualized using a Bio-Rad ChemiDoc Imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).