Hrp conjugated affinipure goat anti mouse igg

The protein levels were determined by the western blotting assay. Total protein lysis was prepared using the RIPA Lysis Buffer (P0013B, Beyotime) and quantified by the BCA Protein Assay Kit (T9300A, Takara). The protein samples for western blotting were prepared using SDS-PAGE Sample Loading Buffer (P0015L, Beyotime). Equal amounts of total proteins were loaded onto 10% SDS-PAGE (P0012AC, Beyotime) and separated by electrophoresis. The separated proteins were transferred onto a PVDF membrane (IPVH00010, Millipore, Shanghai, China) and blocked by 5% skim milk (232100, BD Bioscience, San Jose, CA, USA). The membranes were incubated with primary antibodies Bcl-2 Rabbit Polyclonal Antibody (1:1000, AF0060, Beyotime), Anti-Survivin Rabbit pAb (1:1000, GB11177, Servicebio, Wuhan, China) and GAPDH Mouse Monoclonal antibody (1:5000, 60004-1-Ig, Proteintech, Wuhan, China) at 4 °C overnight. Then HRP-conjugated Affinipure Goat Anti-Mouse IgG (1:5000, SA00001-1, Proteintech) and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (1:5000, SA00001-2, Proteintech) were used to probe the membrane at room temperature for 1 h. The protein bands were visualized using Amersham ECL Prime Western Blotting Detection Reagent (RPN2232, GE Healthcare, Princeton, NJ, USA) and imaged by Tanon-5200 chemiluminescence detection system (Tanon).

OFTu cells were infected with ORFV (MOI = 10) for 36–60 h or treated with chemical activators or inhibitors and then infected with ORFV (MOI = 10) for 60 h and harvested. Cell lysates were prepared by using RIPA lysis buffer (Beyotime) with 1 mM PMSF and quantified with a Pierce BCA protein assay kit (Thermo, Marina, CA, USA). Total protein was resolved by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with blot-blocking buffer (NCM Biotech, Suzhou, China) and incubated with primary antibodies. Antibodies against the following proteins were used in this study: LC3B (Sigma, St. Louis, MO, USA), SQSTM1/P62 (CST, Beverly, MA, USA), phosphorylated and nonphosphorylated mTOR, TSC2, AMPK, AKT, PI3K, ERK1/2, p70S6K (CST), Rheb and GAPDH. In addition HRP-conjugated AffiniPure goat anti-rabbit IgG and HRP-conjugated AffiniPure goat anti-mouse IgG (Proteintech Group, Inc. Rosemont, IL, USA) were used. The blots were visualized with an enhanced ECL chemiluminescent substrate (Biosharp, Beijing, China) in a chemiluminescence imaging system (Tanon, Shanghai, China) after being washed and incubated with secondary antibody, and the expression of the proteins was quantified using ImageJ.

Western blot was performed after cells were lysed using RIPA buffer (Boster, Wuhan, AR0105), and PVDF membranes were used to transfer the proteins. The membranes were blocked and incubated with primary and secondary antibodies. Then, the images were captured by an image capture system (Bio‐Rad, California, ChemiDoc MP). GAPDH and β‐actin were used for normalization. The primary antibodies are listed in Table S2 (Supporting Information). HRP‐conjugated Affinipure goat antirabbit IgG (SA00001‐2, Proteintech, 1:10000) and HRP‐conjugated Affinipure goat antimouse IgG (SA00001‐1, Proteintech, 1:1000) were used as secondary antibodies.