Ipvh85r

GC tissues or cells were collected, and the total protein was extracted by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration was determined using a bicinchoninic acid (BCA) kit (20201ES76, Yeasen Biotechnology, Shanghai, China). Quantification was performed according to different concentrations. Following separation with the use of polyacrylamide gel electrophoresis, the protein was transferred onto a polyvinylidene fluoride membrane (IPVH85R, Millipore, Darmstadt, Germany), which was then blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. Next, the membrane was incubated with primary rabbit antibodies (Abcam, Cambridge, UK) to CXXC4 (ab105400, 1:100), ERK (ab184699, 1:10,000), phosphorylated ERK (ab79483, 1:1,000), PD-L1 (ab233482, 1:100), and GAPDH (ab128915, 1:10,000) overnight at 4°C. The following day, the membrane was cultured with horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (ab205718, 1:20,000, Abcam, Cambridge, UK) for 1 h at room temperature. The membrane was visualized using the developing solution. Protein quantitative analysis was conducted using ImageJ 1.48u software (National Institutes of Health) and expressed as the gray value ratio of each protein to the internal reference GAPDH.

The total protein content was extracted from cells after different treatment protocols using the radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) before estimation of the protein concentration using a bicinchoninic acid (BCA) kit (20201ES76, Yeasen). Subsequent to separation by polyacrylamide gel electrophoresis, the protein was electro-blotted to a polyvinylidene fluoride membrane (IPVH85R, Millipore) by the wet transfer method, which subsequently underwent 1-h 5% bovine serum albumin sealing at ambient temperature. The membrane was probed with primary rabbit antibodies against NLRP3 (ab214185, 1: 1000, Abcam), Cleaved Caspase-1 (#4199, 1: 1000, CST, Danvers, MA, USA), mouse IL-1β (#12242, 1: 1000, CST), and β-actin (ab8226, 1: 3000, CST) overnight at 4 °C. The membrane was supplemented with the corresponding HRP-labeled goat anti-rabbit IgG (ab6721, 1: 5000, Abcam) or goat anti-mouse IgG (ab6789, 1: 5000, Abcam) for 1 h at ambient temperature. The membrane was supplemented with the luminescent solution prior to development. Protein quantitative analysis was implemented using the ImageJ software (National Institutes of Health, Bethesda, Maryland, USA), and the protein quantitative analysis was estimated as the ratio between the gray value of each protein and the gray ratio of the internal reference β-actin.

Total protein extracts were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (IPVH85R, Millipore, MA, USA). After 1 h of blocking with 5% BSA, the membrane was incubated with the corresponding primary antibodies, rabbit monoclonal antibody VWF (ab174290, 1 : 1000, Abcam, Cambridge, UK) and GAPDH (internal reference, ab181602, 1 : 10000, Abcam). After that, the membrane was incubated with HRP-labeled secondary antibody IgG (ab6721, 1 : 5000, Abcam) for 2 h. The signal was detected by the ECL kit (Bio-Rad). The quantitative analysis of protein was carried out by ImageJ software (V1.48, National Institutes of Health, USA).

Porcine satellite cells or muscle tissues were lysed in RIPA buffer containing 1% (v/v) phenylmethylsulfonyl fluoride (PMSF) (ST505, Beyotime, China) to acquired total protein. Approximately 30 μg of protein of each sample was loaded on SDS-PAGE and transferred to PVDF membranes (IPVH85R, Millipore, United States). After blocking with 5% non-fat milk, the proteins in membranes were subjected to immunoblotting analysis with primary and secondary antibodies. Antibodies used in this study were as follows: anti-H3K27me3 (17-622, Millipore, United States), anti-beta-tubulin (GB11017, Servicebio, China), anti-MYOD (sc-760, Santa Cruz Biotechnology, United States), anti-MYH4 (A15293, ABclonal, United States), and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (SA00001-2, Proteintech, United States). The membranes were developed with ECL (WBULS0500, Millipore, United States) for visualization. ImageJ software was used to determine the bands’ signal intensities. The density value of each band was normalized by corresponding beta-tubulin density value.

RIPA lysis buffer (Beyotime) was adopted for total protein content extraction, with protein concentration assessed using BCA kits. Next, the proteins were separated by SDS-PAGE, and then transferred onto PVDF membrane (IPVH85R, Millipore, Darmstadt, Germany). After blocking, the membranes were incubated with specific primary antibodies, rabbit anti-human GLS (ab260047, dilution ratio of 1: 250, Abcam), GDH (ab170895, dilution ratio of 1: 100, Abcam), FBXO4 (ab230302, dilution ratio of 1:1000, Abcam), and GAPDH (ab181602, dilution ratio of 1:10,000, Abcam) at 4 °C overnight. The following day, the membranes were incubated with HRP-labeled IgG (goat anti-rabbit, ab205718, dilution ratio of 1: 5000, Abcam) for 1 h at room temperature. ECL was adopted to visualize the results, with band intensities quantified using the ImageJ software (1.48 u, National Institutes of Health, Bethesda, Maryland). Relative protein expression = gray values of the target protein band / gray values of internal reference GAPDH band.

Cells were collected by centrifugation, washed in PBS, and lysed in RIPA buffer containing protease and phosphatase inhibitors (Thermo 78442) with vortex vigorously. Lysates were then centrifuged at 11,000g at 4 °C for 10 min and the supernatant (quantify the concentration first) was transferred into 4× diluted Laemmli sample buffer containing 2-mercaptoethanol and boiled in 100 °C for 5 min. After that, lysates were separated on 10% Bis-Tris polyacrylamide gels and transferred to PVDF membrane (Millipore IPVH85R). The blots were incubated with primary antibodies overnight at 4 °C and then with secondary antibodies. Blots were developed with the Western Lightening® Plus ECL kit (PerkinElmer). The primary antibodies including anti-Ppp2r1a (Genetex GTX-102206, 1:500), anti-phospho Tyr307 PP2AC (Santa Cruz sc-271903, 1:100), anti-Runx2 (Cell Signaling 8486, 1:1000), anti-human phospho Ser451 Runx2 (Bioss bs-5685, 1:300), anti-PPARγ (ABclonal A0270, 1:500), anti-phospho Ser492 BRD4 (Millipore ABE1451, 1:500), anti-Osterix (Bioss bs-1110, 1:300), anti-collagen X (Abcam ab58632, 1:100), anti-MMP13 (Genetex GTX-100665, 1:500) and anti-GAPDH (Genetex GTX-100118, 1:5000) were used. Immunoprecipitation was performed by Capturem™ IP & Co-IP Kit (Takara, 635721) and the results were analyzed with blotting the elution by anti- Ppp2r1a and anti- human phospho Ser451 Runx2.

Total protein extracts were separated by polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (IPVH85R, Millipore, Germany). The membrane was blocked with 5% bovine serum albumin and probed overnight at 4℃ with primary antibodies against PKP3 (18338-1-AP, 1:1000, Proteintech), ERCC1 (PA5-79217, 1:1000, Invitrogen), Bcl-2 (ab194583, 1:1000, Abcam, Cambridge, UK), Bax (ab32503, 1:1000, Abcam), Total-Cap3 (ab184787, 1:1000, Abcam), Cleaved Caspase-3 (9661, 1:1000, Cell Signaling Technologies [CST], Beverly, MA, USA), ERK1/2 (ab184699, 1:1000, Abcam), phospho-ERK1/2 (ab201015, 1:400, Abcam), p38 (14064-1-AP, 1:1000, Proteintech), phospho-p38 (4511, 1:1000, CST), and β-actin (ab8226, 1:5000, Abcam, serving as the loading control). The membrane was re-probed with the horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit IgG (ab6721, Abcam) or goat anti-mouse IgG (ab6789, Abcam) at room temperature for 1 h. The immunocomplexes on the membrane were visualized using luminescent liquid (1705062, Bio-Rad, California, USA) on Image Quant LAS 4000 C gel imager (GE Healthcare, Chicago, Illinois, USA) and band intensities were assayed using Image J software (National Institutes of Health).