Hrp conjugated anti rabbit igg

Puromycin and G-418 were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). AG1478, and U0126 were purchased from Selleck. The protein A/G agarose and EGF were purchased from Sigma (St louis, MO, USA). The following antibodies were used: anti-β-actin (#8457), anti-cleaved-caspase-3 (#9664), ani-caspase-3 (#9662), anti-cleaved PARP (#5625), anti-EGFR (#4267), anti-Erk1/2 (#4695), anti-phospho-Erk1/2 (#4370), anti-p38 (#8690), anti-phospho-p38 (#9211), anti-AKT(#9272), anti-phospho-AKT (Ser473) (#4060), anti-phospho-JNK (Thr183/Tyr185) (#9251), anti-JNK (#9252), anti-HA-Tag (#3724), anti-c-Cbl (#2747), anti-ubiquitin (P4D1) (#3936) from Cell Signaling Technology (Beverly, MA, USA); anti-Flag-Tag from Sigma-Aldrich (St Louis, MO, USA), anti-GAPDH (AC002), anti-phospho-EGFR (Y1068) (AP0820) from Abclonal Technology(Shanghai, China); anti-14-3-3σ (ab14123), anti-Bim (ab7888) from Abcam (Cambridge, MA, USA); anti-Bcl-2 (12789-1-AP) from proteintech (Chicago, IL, USA); Alexa Flour 488-conjugated anti- rabbit IgG, Alexa Flour 555-conjugated anti-rabbit IgG and Alexa Flour 555-conjugated anti-mouse IgG from Thermo Scientific (Rockford, IL, USA). HRP conjugated anti-rabbit IgG, HRP conjugated anti-mouse IgG from Beyotime Institute of Biotechnology; APC anti-human EGFR antibody (#352905) used for cell surface immunofluorescence staining from BioLegend (San Diego, CA).

The antibodies used were MyoG (ab124800, Abcam), MyoD (ab133627, Abcam), p-ERK1/2 (9101, Cell Signaling Technology), GAPDH (ab8245, Abcam), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (A0208; Beyotime). Total protein from cultured MuSCs was extracted using the Total Protein Extraction Kit (Beyotime) and quantified using the BCA Protein Quantitation Kit (Beyotime) according to the manufacturer’s instructions. Briefly, proteins (~20 μg per sample) were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to a PVDF membrane (Millipore, Bedford, MA, USA), and blocked with blocking buffer (Beyotime) for 1 h at room temperature. The membranes were sequentially incubated with primary anti-mouse MyoG (1:1000), MyoD (1:1000) and p-ERK1/2 (1:1000), washed three times with PBST (0.1% Tween 20 in PBS), and incubated with the secondary antibody conjugated with HRP (1:4000) for 90 min. The membranes were washed three with PBST. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (BeyoECL Plus, Beyotime) and the ChemiDoc XRS⁺ system (Bio-Rad, Hercules, CA, USA). GAPDH (1:2000) served as a loading control.

A Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). The antibodies against phospho-Akt (Ser473), Akt, phospho-JNK (Thr183/Tyr185), JNK, phospho-p38, p38, phospho-NF-κB (Ser536), NF-κB, phospho-IκBα and IκBα were purchased from Cell Signaling Technology (Beverly, MA, USA). Phospho-ERK1/2, ERK1/2, caspase-3, caspase-8, caspase-9, CytoC and GAPDH antibodies were purchased from Abcam (Cambridge, MA, USA). Beta-Tubulin, HRP-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Beyotime (Jiangsu, China). LY294002 (PI3K/Akt inhibitor), PD98059 (ERK1/2 inhibitor), SP600125 (JNK inhibitor) and Bay11-7082 (NF-κB inhibitor) were purchased from Selleck (Houston, USA). FITC-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG were purchased from EarthOx Life Sciences (Millbrae, CA, USA). The 4’,6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (Jiangsu, China).

Ileum samples were lysed on ice in RIPA lysis buffer (Beyotime) supplemented with 1 mM phenylmethylsulfonyl fluoride (Beyotime). The protein concentration was measured using BCA Protine Assay Kit (Beyotime). Protein were separated by 8% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore). Then, membrane was blocked with 5% non-fat milk and incubated with primary antibodies overnight at 4°C and secondary antibodies for 1.5 h at room temperature. Signals were detected with WESTAR NOVA 2.0 (Cyanagen). The antibodies used were: rabbit anti-APN (1:2000, a generous gift from YW Huang¹¹ ), mouse anti-β-actin (1:2000, Beyotime), HRP-conjugated anti-mouse IgG (1:10000, A0216; Beyotime) or HRP-conjugated anti-rabbit IgG (1:10000, A0208; Beyotime).

The SCNs were lysed with a cell lysate containing PMSF and phosphatase inhibitors. The cell lysates were collected and centrifuged at 14,000 rpm for 5 min. The supernatants were taken and the protein concentration was determined using a BCA kit (Beyotime Biotechnology, China). The protein sample was boiled for 10 min before loading, and then electrophoresed using 10% SDS gel, transferred to a PVDF membrane, blocked with 5% skim milk at room temperature for 1 h, and then the membranes were incubated overnight at 4°C with anti-PARP1(Abcam, ab151794, 1:1,000 dilution), anti-PAR(CST, #83732, 1:1,000 dilution), anti-γH2AX(CST, #2577, 1:1,000 dilution), anti-GAPDH(CST, #5174, 1:1,000 dilution), anti-p-AMPK (CST, #2535, 1:1,000 dilution), and anti-AMPK (CST, #2532, 1:1000 dilution) primary antibodies in dilution buffer. The cells were incubated with the horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Beyotime Biotechnology, A0216, 1:1,000 dilution). The membranes were developed using the enhanced chemiluminescence substrate LumiGLO (Millipore, Bedford, MA, USA).