Rabbit anti hdac1

N2a/APP695 cells (1 × 10⁵ cells/mL) were cultured in a 6-well plate for 24 hours and were then treated with RJPs for 24 hours. Cells were washed with cold PBS and lysed with 1 × loading buffer (10 mM Tris, pH 6.8; 1%SDS; 5% glycerin; 0.1 M DTT; bromophenol blue; 1 mM AEBSF (Sigma, USA)). The lysate was collected and boiled at 100 °C for 10 min. Subsequently, an equal volume of sample was electrophoretically separated using 10% SDS-PAGE and then transferred to an NC membrane (Millipore, USA). Membranes were incubated overnight at 4 °C with various primary antibodies. The primary antibodies used in this study: mouseanti-APP (6E10, 1:250, Covance, USA), rabbit-anti-BACE1 (1:500, Abcam, USA), rabbit-anti-HDAC1 (1:5000, AB clonal, Wuhan, China), rabbit-anti-β-actin and rabbit-anti-GAPDH (1:2000, Cell Signaling Technology, USA). The secondary antibodies used were goat-anti-mouse IgG IRDye 800CW (1:10000, Li-COR, USA) and goat-anti-rabbit IgG IRDye 800CW (1:10000, Li-COR, USA). After incubation with the secondary antibody, the membranes were washed 4 times for 5 min each time. Finally, we used the LI-COR Odyssey Infrared Fluorescence Scanning System (Li-COR, USA) to quantify the fluorescence intensity. The intensity of the bands was analyzed using the system 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.