Hrp conjugated goat anti mouse igg

The reagents used in this study include erianin (≥98% purity) (Tauto Biotech, Shanghai, China); 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazolium bromide (MTT) (Biofroxx, Einhausen, German); Annexin V-FITC/PI staining kit (BestBio, Shanghai, China); 2’,7’-Dichlorofluorescin diacetate (DCFH-DA) (Sigma, St. Louis, MO, USA); bicinchoninic acid (BCA) protein assay kit (Sangon Biotech, Shanghai, China); N-Acetyl-L-cysteine (NAC) (Beyotime Biotechnology, Shanghai, China); antibodies against cleaved PARP, cleaved caspase-3, AKT, phosphorylated-AKT (p-AKT), mTOR, phosphorylated-mTOR (p-mTOR), JNK, phosphorylated-JNK (p-JNK), c-Jun and phosphorylated-c-Jun (p-c-Jun) (Cell Signal Technology, Boston, USA); antibodies against β-actin, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (ZSGB-BIO, Beijing, China). Erianin was dissolved in dimethyl sulfoxide (DMSO) (MP Biomedicals, Solon, OH, USA) in a 100 mM stock solution and stored in the dark at −20 °C.

The cells were lysed within RIPA buffer that contained 1% PMSF, and the proteins were loaded onto SDS‐PAGE microgels and transferred to PVDF membranes. Then 50 g/L skim milk powder was managed for blockage, and the primary antibodies diluted in 50 g/L BSA were added, including rabbit anti‐human c‐met (1: 100, Abjent, USA), mouse anti‐human E‐cadherin (1:3000, Abcam, Cambridge, MA, USA), mouse anti‐human N‐cadherin (1:8000, Abcam, Cambridge, MA), mouse anti‐human vimentin (1:1000, Cell Signaling Technology, Danvers, MA, USA) and mouse anti‐rat α‐SMA monoclonal antibody (1:300, Boster, Wuhan, China). They were placed at 4°C for overnight before incubating the blots with HRP‐conjugated goat anti‐mouse IgG (1:500, ZSGB‐BIO, Beijing, China). Darkroom visualization was achieved using ECL substrate visualization signals (Millipore, Billerica, MA, USA), and GAPDH was used as normalized endogenous white matter.

Proteins were extracted from the cells for western blot analysis. In total, 50 μg protein was subjected to 12% SDS-PAGE, transferred to a PVDF membrane (Millipore, Billerica, MA, USA) and incubated with the following antibodies: S100A14 antibody (1:500, Proteintech), Pepsin C antibody (1:500, LifeSpan BioSciences), E-cadherin antibody (1:500, BD Transduction), Orai1 antibody (1:500, Abcam), STIM1 antibody (1:500, Abcam), FAK antibody (1:500, CST, Beverly, MA, USA), MMP2 antibody (1:1000, Abcam), MMP9 antibody (1:1000, Abcam) and MMP11 antibody (1:500, BGI-GBI Biotech). The membrane was then incubated with HRP-conjugated goat anti-mouse IgG (1:2000, ZSGB-BIO) or HRP-conjugated goat anti-rabbit IgG (1:2000, ZSGB-BIO). β-Actin antibody (1:10 000, Sigma, St. Louis, MO, USA) was used as a control. The protein level was detected by ECL (GE Healthcare, Buckinghamshire, UK). Chemiluminescence was visualized using a SmartChemi image analysis system (Sagecreation, Beijing, China).

For Western blot analysis, whole-cell extracts were harvested using RIPA lysis buffer (strong) (CWBIO, Beijing, China) containing protease inhibitor cocktail (CWBIO, Beijing, China) and phosphatase inhibitor cocktail (CWBIO, Beijing, China) at 30 min, 60 min, and 120 min postinfection, separated by SDS–PAGE and transferred onto PVDF membranes (Millipore, Darmstadt, Germany). Subsequently, the membranes were incubated with primary antibodies against monoclonal mouse anti-β-actin (1:4000, TransGen, China), rabbit anti-p-p44/42 MAPK (1:1000, CST, USA), rabbit anti-p-SAPK/JNK (1:1000, CST, USA) or rabbit anti-p-p38 (1:1000, CST, USA) overnight, followed by incubation with HRP-conjugated goat anti-rabbit IgG (1:1000, Beyotime, China) or HRP-conjugated goat anti-mouse IgG (1:4000, ZSGB-BIO, China). Finally, the bands were visualized using Amersham^® Hyperfilm^® ECL™ and MP Autoradiography Films (GE Healthcare).

Ninety-six-well microtiter plates were coated with the purified Nsp10 protein in 0.1 M carbonate- bicarbonate buffer (pH 9.6) at 4 °C overnight and then blocked with 5% skim milk for 1 h at 37 °C. After washing three times with 0.1% Tween-20 in phosphate buffered saline (PBS), the plates were incubated with 100 μl of hybridoma supernatants at 37 °C for 1 h. Subsequently, the plates were washed thrice and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZSGB-BIO, China) at a dilution of 1:10,000 in PBS at 37 °C for 1 h. A substrate solution containing o-phenylenediamine (OPD) was added for color development and the enzymatic reaction was stopped with 2 M H₂SO₄. The absorbance was measured at 490 nm using a Microplate Reader.

The transfected HEK293T cells in 24-well-plate were lysated with 200 μl Protein lysis buffer (50 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate, 0.1% Bromophenol blue, 10% glycerol, 1% β-Mercaptoethanol), denatured for 10 min at 95 °C before loading. Using a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel to separate proteins, samples were transferred to a PVDF membrane (Millipore, #IPVH00010). The blotted membranes were blocked with blocking solution containing 5% non-fat milk powder (Sangon Biotech, A600669) in PBS-T (0.1% Tween-20 in PBS buffer) for 60 min, followed by an overnight exposure at 4 °C in the same buffer to the following primary antibodies: (a) rabbit anti-mCherry polyclonal antibody (1:5000, Millipore #AB356482); (b) rabbit anti-GFP polyclonal antibody (1:2000, Proteintech 50430-2-AP); (c) mouse anti-β-Tubulin monoclonal antibody (1:10,000, ProteinTech # 66240-1-Ig). The membranes were washed and incubated for 2 h at room temperature with the corresponding secondary antibodies: (a) HRP-conjugated goat anti-rabbit IgG (1:10,000, ZSGB-Bio #ZB-5301); (b) HRP-conjugated goat anti-mouse IgG (1:10,000, ZSGB-Bio #ZB-5305). Signals were detected and visualized by exposure to Clarity Western ECL Substrate (BioRad, #1705061) and digitized with Tanon 5500 chemiluminescence imaging analysis system. The uncropped scans of blots are provided as a Source Data file.