Mouse anti histone h3

Cells were lysed with RIPA buffer, and the protein concentrations were determined by BCA assay (Solarbio, #9c0020, Beijing, China). Total protein (40 μg) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% gel and electrotransferred to polyvinylidene fluoride membranes. The membranes were incubated overnight at 4°C with rabbit monoclonal anti-hnRNP C1+C2 (Abcam, ab133607, Cambridge, England), and anti-LC3A/B, anti-ATG3, anti-ATG5, anti-ATG7, and anti-BECLIN1 (for autophagy detection, Abcam, ab133607, Autophagy Antibody Sampler Kit 4445, England). Alternatively, blots were incubated with rabbit polyclonal anti-14-3-3ε (Abcam, ab43057, Cambridge, England), anti-GAPDH (ABclonal, A10868, Wuhan, China), mouse anti-histone H3 (Beyotime, AF0009, Shanghai, China). Blots were then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Later, they were probed using the ECL Reagent and visualized by a Western blotting analysis detection system. Relative quantitative measurement of Western blotting was performed using ImageJ software.

The IPEC-J2 cells infected with PEDV at appropriate MOIs or transfected with recombinant expression plasmids or siRNA were used for total protein extraction using the cell lysis buffer (Beyotime, China). The protein concentration of the whole-cell lysates in different treatments was measured by a bicinchoninic acid (BCA) protein assay kit (Thermo). Afterward, the heat-denatured protein samples were loaded onto 12% SDS-PAGE gels for protein fractionation followed by blotting onto a 0.22-μm polyvinylidene difluoride membrane (PVDF; Millipore). The proteins on the blots were then probed by specific primary and secondary antibodies, including rabbit anti-STAT1 (CST), rabbit anti-phospho-STAT1 at Y701 (ABclonal), rabbit anti-acetyl-lysine (Abcam), rabbit anti-acetyl-H3-K27 (ABclonal), rabbit anti-HDAC1 (ABclonal), rabbit anti-ISG15 (Beyotime), mouse anti-β-actin (Thermo), mouse anti-histone H3 (Beyotime), rabbit anti-HA tag (CST), mouse anti-PEDV N (monoclonal antibody reserved in our laboratory), goat anti-mouse IgG (Invitrogen) and goat anti-rabbit IgG (Invitrogen). The target protein bands on the membrane were developed using an ECL kit (Cyanagen, Italy) and were visualized by an imaging system (SageCreation, Beijing, China) for densitometric analysis of their relative levels of expression.