Anti β actin

Liver samples or hepatocyes were lysed with RIPA peptide lysis buffer (Beyotime Biotechnology, Jiangsu, China) containing 1% protease inhibitors (Pierce) and Western blotting analysis were performed according to standard procedures. Primary antibodies were used as follows: anti‐Pknox1 (1:1000, Novus, USA), anti‐IR (1:1000; Abcam, Cambridge, UK), anti‐pY‐IRS1 (1:1000; Abcam), anti‐IRS1 (1:2000; Abcam), anti‐pS‐IRS1 (1:2000; Abcam), anti‐pY‐IRS1 (1:1000; Abcam), anti‐AKT (1:1000; Cell Signaling Technology, MA, USA), anti‐pY‐AKT (1:1000; Cell Signaling Technology), and anti‐β‐actin (1:3000, Huabio, China). Protein bands were developed using the Enhanced Chemiluminescence (ECL) system and were visualized by using the ChemiScope Western Blot Imaging System (Clinx Science Instruments Co., Ltd). The gray‐scale value assay was performed by using Image J software (Rawak Software, Inc. Germany).

The sequences of the primer pairs used in qPCR are as follows: Sag-Fwd: 5′-TG GAGGACGGCGAGGAAC-3′, Sag-Rev: 5′-CCCCAGACCACAACACAGTC-3′; Rbx1-Fwd: 5′-CTTTGTATCGAATGTCAGGC-3′, Rbx1-Rev: 5′-GTCACTAGACGAGTAACAG-3′; Ube2f-Fwd: 5′-GACTGTAAGCCCAGATGAG-3′, Ube2f-Rev: 5′-CCTTTAATGTTCTAGTGGG-3′; Ube2m-Fwd: 5’-CTGTCCTGATGAAGGCTTC-3′, Ube2m-Rev: 5′-GTTCGGCTCCAAGAAGAG-3′; Gapdh-Fwd: 5′-GCCGCCTGGAGAAACCTGCC-3′, Gapdh-Rev: 5′-GGTGGAAGAGTGGGAGTTGC-3′.
The antibodies used in western blot are as follows: anti-Ube2f, Proteintech, 17056-1-AP (dilution 1: 1000); and anti-β-Actin, HuaBio, M1210-2 (dilution 1: 5000).

Protein extracts were sampled from the zebrafish hypothalamus and separated by 10% SDS-PAGE (ACE Biotechnology, China) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, United States). The membranes were blocked with QuickBlock™ Blocking Buffer (Beyotime, China) at room temperature, and incubated with following the primary Abs overnight at 4°C: rabbit polyclonal anti-Gαq (ABclonal, China) and rabbit polyclonal anti-β-actin (HUABIO, China). The blots were detected with HRP-conjugated anti-rabbit IgG (1:2000) and visualized by an enhanced chemiluminescence (ECL) reagent (Beyotime, China).

Total proteins were extracted using lysis buffer consisting of 2% sodium dodecyl sulfate (SDS), 1% Triton X-100, 50 mM Tris-HCl, and 150 mM NaCl (pH 7.5), separated by 10% SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was then blocked with 5% skim milk and incubated with primary antibodies at 4°C overnight. After hybridization with horseradish peroxidase (HRP)-conjugated secondary antibodies, the membrane was visualized using enhanced chemiluminescence (ECL) reagents (Bio-Rad, Hercules, CA, USA), and a Quantity One system (Bio-Rad, USA) was applied for analysis. The primary antibodies were anti-myc (Huabio, China), anti-TNFAIP3 (Proteintech, USA), anti-β-actin (Huabio, China), anti-caspase-3 (Cell Signaling Technology [CST], USA), anti-AGO2 (Abcam, USA), and anti-PDCoV N protein (Medgene Labs, USA), and the anti-PDCoV S protein polyclonal antibody was stored in our lab.

Cell or tissue lysate was resuspended in 5× SDS loading buffer, subsequently incubated at 100°C for 5 min and centrifuged at 12,000 × g for 10 min. Protein concentrations were detected using a BCA Protein Assay Kit (Thermo Fisher Scientific). A total of 20 μg of protein from the tissue or cell lysate was separated by SDS-PAGE gel (Thermo Fisher Scientific) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membrane was blocked using 5% nonfat milk for 2 h at room temperature and then incubated with appropriate primary antibodies: anti-NLRP3 (Abclonal, Wuhan, China) (1 : 1,000) and anti-Caspase-1 (Abclonal, Wuhan, China) (1 : 1,000) in blocking buffer overnight at 4°C. Anti-β-actin (HuaBio, Shanghai, China) (1 : 2,000) was used as a loading control. After washing three times with PBST, the membranes were incubated with HRP-conjugated secondary antibodies for 1.5 h at room temperature. The bands were detected using an ECL kit (MultiSciences, Hangzhou, Zhejiang, China).

Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China, P0013B). Total protein was quantified by BCA protein assay (Bio‐Rad, Berkery). Each sample was adjusted to equal amount of protein using 5X loading buffer for loading. The samples were separated by SDS‐PAGE, transferred to a polyvinylidene fluoride membrane, and immunoblotted with the following antibodies: GDF11 (DGDF80, R&D Systems,Emeryville, CA,USA), HGF (ab83760, Abcam, USA), VEGFR1 (ET1605‐11,Huabio，China), CD31 (#77699, Cell Signalling TechnologyBoston,MA, USA), VEGFR2 (#9698, following Abs are all from Cell Signalling Technology,USA), phospho‐p44/42 (Thr202/Try204 phospho‐ERK1/2,#4370), p44/42 MAPK (Erk1/2,#4696), BCL2 (#2827), BAX (#14796), Cleaved Caspase3 (#9661), phospho‐Smad2 (Ser465/Ser467,#18338), phospho‐Smad3 (Ser423/425, # 9520), Smad2(#5339), Smad3(#9523), anti‐β‐actin (#3700), EIF4E (R1512‐8,Huabio, China), Phospho‐eIF4E (S209) (ET1608‐66,Huabio, China) and VEGF ( ER30607,Huabio, China),at 4°C overnight. After incubation of the membranes with peroxidase‐conjugated secondary antibodies (Cell Signalling Technology,USA), bands were visualized using enhanced chemiluminescence reagents (Bio‐Rad,Los Angeles, CA,USA).