Ab49900

Freshly harvested cells were lysed in RIPA buffer. Protein concentrations were determined by Pierce BCA^™ Protein Assay Kit (Pierce). Proteins were separated via Mini Protean TGX stain free gel 4–15% (BioRad) and transferred to polyvinylidene difluoride membrane by using iBlot 2 PVDF Regular Stacks (Invitrogen) and iBlot system transfer system (LifeTechnologies). Membranes were blocked in 5% BSA solution (Sigma). Primary antibodies were diluted following the manufacturer’s instructions: anti-beta Actin, [AC15] (HRP-conjugated) ab 49900, Abcam (1:25000) and antiPTGR1 [EPR13451–10], ab181131, Abcam (1:1000). Signals were developed using Clarity Western ECL Substrate (BioRad) and Image Quant LAS4000 System (GEHealthCare).

Cells were lyzed with lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP40) containing Complete Protease Inhibitor Cocktail (Roche). The cell lysates were separated by standard SDS-PAGE (e-PAGEL, ATTO, Tokyo, Japan) and analyzed by immunoblotting. The following antibodies were used for immunoblotting: anti-PD-L1 (ab213480, Abcam, Cambridge, UK), anti-EGR1 (ab133695, Abcam), anti-c-Myc antibody (Y69) (ab32072, Abcam), goat anti-rabbit IgG (H+L)-HRP Conjugate (Bio-Rad), anti-phospho-p38 antibody (Thr 180/Tyr 182; sc-17852-R, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-SAPK/JNK (Thr183/Tyr185; 81E11, Cell Signaling Technology, Danvers, MA, USA), anti-IκBα mouse antibody (L35A5, Cell Signaling Technology), anti-phospho-IκBα (Ser32/36; 5A5, Cell Signaling Technology), anti-β-actin (C4) (sc-47778, Santa Cruz Biotechnology), and anti-β-actin-HRP (AC-15) (ab49900, Abcam). The Western HRP Substrate (Pierce ECL Western Blotting Substrate, Thermo Fisher Scientific) was used for the development of positive signals, and chemiluminescence was detected using a ChemiDoc XRS+ System (Bio-Rad, Hercules, CA, USA).

Cells were washed with 1X PBS (162528, MP Biomedicals) and lysed with 1X Laemmli buffer (1610747, BIO-RAD). Cell lysates were loaded and resolved using a 10% SDS-PAGE gel, and the separated proteins were transferred onto a PVDF membrane (IPVH00010, Immobilon-P; Merck). Blocking was performed using 5% Skimmed milk (70166, Sigma-Aldrich) in 1X PBS containing 0.05% Tween 20 (P1379, Sigma-Aldrich) (1X PBST) for 2 hr at room temperature with slow rocking. Primary antibody incubation was performed overnight (12 hr) at 4°C using SARS-CoV / SARS-CoV-2 (COVID-19) spike antibody (180 KDa) (GTX632604, GeneTex, RRID: AB_2864418 or NR-52947, BEI Resources, NIAID, NIH). Secondary antibody incubation was performed for 2 hr at room temperature with slow rocking using Goat Anti-Mouse IgG H&L (ab6789, Abcam, RRID: AB_955439) or Goat Anti-Rabbit IgG H&L (ab6721, Abcam, RRID:AB_955447). The blots were developed using Clarity Western ECL Substrate (1705061, BIO-RAD). Blots were probed for beta-actin (42 KDa) using mouse monoclonal antibody to beta Actin [AC-15] (HRP) (ab49900, Abcam, RRID: AB_867494). All antibodies were authenticated by the respective companies and relevant documentation is available in Supplemental Data.

Cells were prepared for western blotting in the following manner, and all the steps were conducted at 4°C. Cells, treated with inhibitors or DMSO in fresh media, (changed 3 h before treatment) were washed once with cold PBS, collected in cold PBS, centrifuged and lysed in RIPA buffer (25 mM Tris, pH 8.0, 1% NP40, 0.5% DOC, 0.1% SDS, 150 mM NaCl) supplemented with cOmplete™ protease inhibitor cocktail (Sigma), PhosSTOP™ (Sigma), and 50 μM thiamet-G (Sigma). After this, samples were loaded on an SDS-PAGE gel and transferred to nitrocellulose membrane for immunoblotting. Antibodies used in this study include: anti-OGT (24083S, CST), anti-O-GlcNAc (RL2, ab2739, Abcam) and anti-actin (ab49900, Abcam).

Cells were lysed in ice‐cold radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with phosphatase and protease inhibitors, and protein concentration was determined using the DC Protein assay (Bio‐Rad). Equal amounts of proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Sigma) for 1 h at 400 mA. Membranes were blocked in 5% milk and incubated with antibodies against CPEB4 (Mouse Monoclonal ERE149C, 1:500, homemade), β‐Actin‐HRP (1:15,000, ab49900, Abcam), vinculin (1:1,000, ab130007 Abcam), DDIT4 (1:1,000, 10,638‐1‐AP, Proteintech), HERPUD2 (1:500, sc‐398583, Santa Cruz), phospho‐eiF2α (s51) (1:1,000, ab32157, Abcam), total eiF2α (1:1,000, 9722S, Cell Signaling), CHOP (1:1,000, CST5554T, Cell Signaling), and BiP (1:1,000, C50B12, Cell Signaling).