Anti muc16

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.

HCECs were lysed in Pierce RIPA Buffer (no. 89901; ThermoFisher Scientific) containing a 1× Halt Protease and Phosphatase Inhibitor Cocktail (no. 78440; ThermoFisher Scientific). The protein concentration was measured by a Pierce Rapid Gold BCA Protein Assay Kit (no. A53226; ThermoFisher Scientific). An equal amount of protein was boiled in a T-Pro Laemmli (SDS sample) Reagent (no. JB06-F002; T-Pro Biotechnology, Zhonghe, Taiwan), separated by 10% SDS/PAGE, and transferred onto Amersham Hybond P Western blotting membranes, PVDF (no. 10600023; GE Healthcare Life Sciences, Chicago, IL, USA). The membranes were incubated for 1 h in a blocking buffer (Tris-buffered saline solution [TBS] and 5% BSA) and then probed by overnight incubation at 4°C with either anti-MUC16 (no. SC-365002; Santa Cruz Biotechnology, Dallas, TX, USA) or anti-beta-actin (no. ab8226; Abcam) antibody. After being washed in Tris-buffered saline solution–T, the blot was incubated with horseradish peroxidase-conjugated secondary antibodies and detected by the ECL Select Western Blotting Detection Reagent (no. RPN2235; GE Healthcare Life Sciences).