Hrp goat anti mouse igg as003

The antibody against B2 protein of RGNNV was produced by aBIOTECH company (Jinan, China) through immunizing rabbits with B2 protein expressed in E. coli. The affinity and specificity of anti-B2 serum were confirmed by positive band in wtRGNNV-infected SSN-1 cells, but no signal was detected in non-infected SSN-1 cells through Western blotting assay (Figure S1). Other antibodies and reagents used in the current study were mouse anti-β-actin (66009-1, ProteinTech Group, Chicago, IL, USA), horseradish peroxidase (HRP) goat anti-mouse IgG (AS003, Abclonal, Wuhan, China), HRP goat anti-rabbit IgG (AS014, Abclonal, Wuhan, China), Alexa Fluor^® 488 goat anti-mouse IgG (A-11001, Invitrogen, Grand Island, NY, USA), 4′,6-diamidino-2-phenylindole (DAPI) (C1002, Beyotime, Shanghai, China), proteinase inhibitor cocktail (P8340, Sigma, Darmstadt, Germany), phosphatase inhibitor cocktail 3 (P0044, Sigma, Darmstadt, Germany).

Cells were lysed using standard RIPA strong buffer supplemented with 1% PMSF and a proteinase cocktail (Solarbio, China). The protein concentration was determined using a BCA protein assay kit (GK10009, GLPBio). Equal amounts of protein were loaded onto SDS‒PAGE gels, transferred to PVDF membranes, and blocked with 5% skim milk. Specific primary antibodies were applied at 4 °C overnight. A chemiluminescence system (Tanon 5200, Tanon, China) was used for exposure. The following antibodies were used: β-actin (AC038, 1:10,000), P70S6 kinase 1 (A2190, 1:1000), phospho-p70 S6 kinase-T389 (AP1389, 1:1000), HRP goat anti-rabbit IgG (AS014, 1:5000), and HRP goat anti-mouse IgG (AS003, 1:5000) from ABclonal Technology (Wuhan, China); GPX4 (ab231174, 1:1000) and FACL4 (ab155282, 1:1000) from Abcam (Shanghai, China); HMGA2 (#61041, 1:1000) from Active Motif (California, USA); and FLAG (#14793, 1:2000) from Cell Signaling Technology (Shanghai, China).

Mouse anti-p53 (sc-126), mouse anti-BMP2 (sc-137087), mouse anti-PolG (sc-390634) and mouse anti-Runx2 (sc-101145) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, US). Rabbit anti-p53 (#10442-1-AP), rabbit anti-Runx2 (#20700-1-AP) and mouse anti-GAPDH (10494-1-AP) were purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Rabbit anti-PolG (#13609) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase (HRP) goat anti-rabbit IgG (AS014), HRP goat anti-mouse IgG (AS003) were purchased from ABclonal Biotech, Co., Ltd. (Wuhan, China). Rabbit anti-alpha Tublin (WL02296) and rabbit anti-COX4 (WL02203) were purchased from Wanleibio, Ltd. (Shenyang, China).