Goat anti mouse igg

The protein concentrations of the cell lysate were determined using the Bradford protein assay kit using the bovine serum albumin as the protein standard (Beyotime). Equal amounts of protein (25 μg) per sample were separated on 10 % SDS polyacrylamide gels and then transferred onto 0·45 μm PVDF membranes (Millipore). The membranes were blocked with a blocking buffer (Beyotime) at room temperature for 2 h, followed by 4 h incubation at room temperature with the primary antibodies mentioned in online Supplementary Table S1. After washing with (TRIS-buffered saline containing 0·02 % (v/v) Tween-20 (TBST) three times, the membranes were then incubated with secondary goat-anti-rabbit IgG (Beyotime, dilution 1:500) or goat-anti-mouse IgG (Beyotime, dilution 1:500) conjugated with horseradish peroxidase in a blocking buffer for 4 h at 37°C. The membranes were then washed with TBST three times and immediately incubated with enhanced chemiluminescence Western blotting substrate kits (Beyotime), followed by visualisation using a chemiluminescence imaging system (CLiNX Science Instrument). The relative intensities of the bands were calculated with ImagePro Plus 6.0 software (Media Cybernetics). The β-actin protein was used as the reference protein.

Approximately 1.2 × 10⁶ of PK-15 cells were lysed in ice-cold cell lysis buffer (50 mM Tris-HCl [pH = 6.8], 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue) supplemented with protease inhibitor (Beyotime) and phenylmethylsulfonyl fluoride (PMSF) (Beyotime), with cell lysates precleared at 4 °C by centrifugation at 15,871 × g for 10 min. The precleared lysates were separated by 10% polyacrylamide gel SDS. Separated proteins were then transferred onto a nitrocellulose membrane and probed with Cas9 (GeneTex, #GTX53807, 1:3000), EMC3 (Santa Cruz Biotechnology, #sc-365903, 1:500), CALR (Abclonal, #A1066, 1:1000) antibody, with β-tubulin (Sungenebiotech, #KM9003T, 1:5000)/GAPDH antibodies (Beyotime, #AF5009, 1:3000) used as an internal loading control. The primary antibodies were detected with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (Abclonal, #AS014, 1:5000) or goat anti-mouse IgG (Beyotime, A0216, 1:1000), and the secondary antibodies were visualized by ECL Prime Western Blotting Detection Reagents (GE Healthcare, UK).

After culture, NP cells seeded on the glass coverslips were fixed with 4% paraformaldehyde for 20 min. Then, they were permeabilized with 0.1% Triton X-100 for 30 s, blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies (aggrecan: Novus, NB600-504; collagen II: Abcam, ab185430) overnight at 4°C. After incubation with goat antimouse IgG or goat anti-rabbit IgG (1:400 dilution, Beyotime, China) for 2 h, positive staining was developed and the cellular nucleus was stained with the hematoxylin solution. Finally, staining intensity was analyzed using the Image-Pro Plus software (Version 5.1, Media Cybernetics, Inc.).

Total protein isolation was carried out from infected cells by employing the phenylmethanesulfonyl fluoride (Beyotime) mixed with RIPA lysis solution (Beyotime). Protein concentration was measured by the BCA Protein Assay Kit (Beyotime). 25 μg protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Merk Millipore, Billerica, MA, USA). After blocked with 5% skimmed milk, membranes were incubated with the antibodies against RUNX1 (1:400; Affinity, Changzhou, China) or β-actin (1:1000; Santa Cruz) at 4 °C overnight and then with the secondary antibodies horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000; Beyotime) or goat anti-mouse IgG (1:5000; Beyotime). Protein blot bands were visualized with WD-9413B gel imaging system (Liuyi biotechnology, Beijing, China).

BxPC-3 and PANC-1 cells treated with Baicalein, inhibitors, or siRNAs were lysed in the RIPA buffer (Beyotime Biotechnology). The concentrations of the total proteins were measured using the Bicinchoninic acid (BCA) method (Beyotime Biotechnology). Protein samples (20 μg) were subjected to SDS-PAGE and transferred to nitrocellulose membranes by electroblotting. The membranes were incubated with the primary antibodies and then the corresponding secondary antibodies (horseradish peroxidase-conjugated goat anti-Rabbit IgG or goat anti-mouse IgG at 1:1000 dilution, Beyotime Biotechnology). The signals were detected using Super ECL Detection Reagent (High-sig ECL Western Blotting Substrate, 180-5001, Tanon, Shanghai, China). Primary antibodies used: NEDD9 (#4044, 1:500), Akt (#4691, 1:1000), p-Akt S-473 (#4060, 1:1000), p-Akt T-308 (#2965, 1:1000), ERK1/2 (#9102, 1:1000), p-ERK1/2 (#9101, 1:1000), PDK1(#3062, 1:1000), P21(#8831, 1:1000), P27(#3686, 1:1000) and cleaved caspase-9 (#7237, 1:1000) from Cell Signaling Technology Inc. (Danvers, MA, USA), Bax (AB026, 1:1000) and Bcl-2 (AB112, 1:1000) from Beyotime Biotechnology (Shanghai, China).

Western blot analysis was undertaken as detailed elsewhere [38 (link)]. Twenty micrograms of total cellular proteins were electrophoresed through 10% bis–Tris SDS–polyacrylamide gels, transferred to a polyvinyl difluoride (PVDF) membrane (Thermo Fisher Scientific, Shanghai, China) and then processed for immunoblotting. The first antibodies used for this investigation were from Proteintech [β-actin (66009-1-Ig), ND1 (19703-1-AP), ND5 (55410-1-AP), NDUFS1 (12444-1-AP), NUDFB8 (14794-1-AP), SDHB (10620-1-AP), COX5A (11448-1-AP), BAX (60267-1-Ig), Caspase 3 (19677-1-AP), and Caspase 9 (10380-1-AP)], ABclonal Technology [ND6 (A17991), NDUFA10 (A10123), NDUFS5 (A1265), NDUFC2 (A15073), NDUFA11 (A16239), NDUFS2 (A12858), Cytochrome C (A4912), SOD1 (A12537), SOD2 (A19576), Catalase (A11220), TOM20(A19403), FLAG (AE005), LC3B (A7198), PINK1 (A7131), Parkin (A0968), and P62 (A11250)], Abcam Biotechnology [ND3 (ab192306)], Abcepta [ND4L (AP17147b)], and Cell Signaling Technology [Bcl-xL (2762S) and Caspase 7 (12827)], respectively. The secondary antibodies used for these assays were from Beyotime Biotechnology (peroxidase AffiniPure goat anti-rabbit IgG [A0208] and goat anti-mouse IgG [A0216]), respectively. The protein signals were detected using the ECL system (MilliporeSigma, Burlington, MA). Quantification of density in each band was performed as detailed previously [38 (link)].

The liver tissues were stored at −80°C. Tissue samples were homogenized in ice-cold RIPA lysis buffer (Millipore, Billerica, MA, USA) for protein extraction. Tissue debris was removed by centrifugation, and the resulting supernatants were collected and analyzed for protein concentration by the BCA protein assay kit. The protein was separated on a 10% SDS polyacrylamide gel and then transferred to nitrocellulose membranes (Beyotime Biotech, Shanghai, China). The membranes were incubated with specific primary antibodies overnight at 4°C. The primary antibodies included anti-mouse β-actin (Beyotime Biotech, Shanghai, China), anti-rabbit ATF6, anti-rabbit CHOP, anti-rabbit Wnt3a, and anti-rabbit β-catenin (Company ABclonal, Inc., Wu Han, China). After washing, the membranes were allowed to react with diluted horseradish peroxidase-conjugated secondary antibodies, including goat anti-rabbit IgG antibody and goat anti-mouse IgG (Beyotime Biotech, Shanghai, China) at room temperature for 2 h. An enhanced chemiluminescence system (Beyotime Biotech, Shanghai, China) was used to visualize antibody-antigen complexes.