A16110

All cells were either mock-infected or infected with the indicated viruses (MOI = 3.0). One day post-infection, the cells were lysed in 1% sodium dodecyl sulfate (SDS). Equivalent lysate volumes were separated on 10% SDS-polyacrylamide gels, then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk dissolved in TBST (20M Tris, 150mM NaCl, 0.1% Tween 20, pH 7.4) for 1 hour and probed with one of the following primary antibodies: anti-PKR (sc-6282; Santa Cruz Biotechnology, Inc.), anti-phospho-PKR (ab32036; Abcam), anti-eIF2α or anti-phospho-eIF2α (Ser51) antibody (9722 and 9721, respectively; Cell Signaling Technology), anti-TRS1 999^{27 (link)}, or anti-actin (A2066; Sigma). All primary antibodies were diluted in TBST containing 5% BSA and incubated overnight at 4°C with the membrane. Membranes were washed with TBST three times for 5 mins and then incubated for 1 hour at room temperature with either donkey anti-rabbit or goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (A16110 or 62–6520, respectively; Invitrogen) at 1:10,000 in TBST containing 5% (w/v) nonfat milk. Proteins were detected using the Amersham chemiluminescent detection system (GE Healthcare) according to the manufacturer’s recommendations. Membranes were imaged using the iBright Imaging System (Invitrogen).

Membrane and cytoplasmic proteins of oocytes were extracted using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific). Equal amounts of protein samples were prepared by adding 2× Laemmli sample buffer (Bio-Rad) and loaded on SDS–polyacrylamide gel electrophoresis gels. After gel electrophoresis, membrane and cytoplasmic proteins were probed by Western blot following standard protocols. KCNQ2 and KCNQ3 proteins were detected with primary antibodies rabbit anti-KCNQ2 (1:1000 dilution; Abcam, ab22897) and rabbit anti-KCNQ3 (1:1000 dilution; Abcam, ab66640), respectively, followed by goat anti-rabbit secondary antibody (1:5000 dilution; Invitrogen, A16110), and visualized using enhanced chemiluminescence (ECL Western Blotting reagent, Thermo Fisher Scientific) and CL-XPosure Film (Thermo Fisher Scientific). β-Actin antibody (1:1000 dilution; Abcam, ab8227) was used to probe for control proteins.

Cells and 100,000 g cell pellets were lysed in RIPA buffer (10mM Tris-HCL, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140mM NaCl) with 1mM PMSF for 15min on ice. Protein was quantified using a Pierce BCA Protein Assay (Thermo Scientific). Lysates were resolved on a 4%–12% BisTris Bolt mini gel (Invitrogen) and transferred to nitrocellulose membrane. Membranes were blocked with 5% powdered milk or 5% BSA and washed with TBS-T (0.1% Tween-20, 137mM NaCl, 20mM Tris Base). Primary antibodies were used at manufacturer recommended dilutions and included anti-Alix (Abcam, EPR15314 ab186429, RRID: AB_2754981), anti-Tsg101 (Abcam, ab30871, RRID: AB_2208084), anti-CD63 (Abcam, EPR21151, ab217345, RRID: AB_2754982), anti-Syntenin (Abcam, ab19903, RRID: AB_445200), anti-Arf6 (Abcam, ab77581, RRID: AB_2058475), and anti-CD9 (Santa Cruz, KMC8.8, sc-18869, RRID: AB_2076043). Secondary HRP antibodies were goat anti-rabbit (Invitrogen, A16110, RRID: AB_2534782) and donkey anti-rat (Invitrogen, A18745, RRID: AB_2535522), and blots detected with Pierce ELC Western Substrate (Thermo) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo) on an AI600 imager (GE Healthcare). Bands were quantified using ImageJ, RRID:SCR_003070.