P smad3 ser423 425

Total protein was extracted from NFPAs or normal pituitaries using lysis buffer (Applygen, Beijing, China) following the manufacturer’s instructions. The protein concentration was measured using the bicinchoninic acid method. Protein samples (30 μg) were separated by electrophoresis using 10% sodium dodecyl sulfate polyacrylamide gels, and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20, and incubated with rabbit antibodies against human Smad2 (1:1000; Cell Signaling Technology, Boston, MA, USA), p-Smad2 (Ser465/467) (1:1000; Cell Signaling Technology, Boston, MA, USA), Smad3 (1:1000; Cell Signaling Technology, Boston, MA, USA), p-Smad3 (Ser423/425) (1:1000; Cell Signaling Technology, Boston, MA, USA), and GAPDH (1:5000; Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the membranes were incubated with the secondary antibody anti-rabbit IgG (1:4000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Finally, the membranes were developed using an ultrasensitive chemiluminescence protein dye detection system (ECL Plus; Amersham Pharmacia Biotech, Piscataway, NJ, USA) and exposed to X-ray films (Kodak, Rochester, NY, USA). The bands were subjected to grayscale scanning and semi-quantitative analysis using Quantity One software (Bio-Rad, Hercules, CA, USA).

Protein extracts from the tibialis anterior and soleus muscles of wild-type and VDR^−/− mice were obtained by homogenizing muscles in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) containing phosphatase inhibitors (10 mM Na₃VO₄, 100 mM NaF) and protease inhibitors (Roche). The proteins were resolved by SDS-PAGE (8%) and transferred onto nitrocellulose membranes. The membranes were incubated with primary antibodies followed by incubation with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies and visualized using Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Blots were probed with antibodies against p-Stat3 (clone # B-7, cat # sc-8059, Santa Cruz Biotechnology), p-rpS6 (clone # 91B2, cat # 4857), S6 ribosomal protein (clone # 5G10, cat # 2217), p-p70S6 kinase (Thre389, #9205, Cell Signaling Technology, Beverly, MA), p70S6 kinase (#9202s, Cell Signaling Technology, Beverly, MA), p-Stat5 (Clone C11C5, cat # 9359), Stat5 (cat # 9363), Stat3 (cat # 4904), IL-6 (cat # 12912, Cell Signaling Technology, Beverly, MA), p-Smad3 (Ser423/425) (C25A9, #9520, Cell Signaling Technology, Beverly, MA), slow myosin (NOQ 7.5.4D, cat # M8421, Sigma-Aldrich), Myogenin (F5D, #ab1835 Abcam), GAPDH, and β-actin (cat # sc-47724 and # sc-47778, respectively, Santa Cruz Biotechnology).

Human recombinant TGF-β1, PDGF and BMP4 were purchased from Biolegend (San Diego, CA). Fast-start universal SYBR Green master mix was from Roche, and sb431542, MG132 and chloroquine were from Sigma. The antibodies used for immunoblotting (IB): GAPDH 1:10000 (Sigma), β-actin 1:5000 (Santa Cruz),Smad2 1:1000 (Cell Signalling), Smad3 1:1000 (Cell Signalling), p-Smad2 (Ser465/467) 1:1000 (Cell Signalling), p-Smad3 (Ser423/425) 1:500 (Cell Signalling), IDH1 1:1000 (Origene), SP1 1:1000 (Sigma), α-tubulin 1:1000 (Santa Cruz), p-p38 1:1000 (Cell Signalling), p38 1:1000 (Cell Signalling), p-Erk1/2 1:2000 (Cell Signalling), Erk1/2 1:1000 (Cell Signalling), S100A4/FSP1 1:500 (Abnova), TGFBRI 1:1000 (Cell Signalling), TGFBRII 1:1000 (Abcam), p-TGFBRI 1:200 (Abcam), Cav1 1:1000 (ProteinTech), FKBP12 1:1000 (ProteinTech), and SARA 1:1000 (ProteinTech).

Tissue or cell proteins were prepared according to published protocols (Liao et al., 2015 (link); Yuan et al., 2016 (link)). Briefly, mouse heart tissue or NRCMs were lysed in RIPA lysis buffer for total protein extraction. BCA Protein Assay Kit was used for quantifying protein concentration. Proteins (50 μg/sample) from different samples were used for SDS-PAGE electrophoresis and subsequently transferred to PVDF membrane (Millipore). After blocking with 5% BSA for 1 h, blots were then incubated with primary antibodies overnight at 4°C. The next day, these blots were incubated with secondary antibody conjugated with peroxidase (1:10,000, the Jackson Laboratory) for 1 h at room temperature. All blots were visualized using ChemiDocTM XRS+ (Bio-Rad). The bands were quantified and analyzed using Image-Pro Plus 6.0. The expression of specific proteins was normalized to corresponding GAPDH before relative quantitative analysis. The primary antibodies used in this study were purchased from Cell Signaling Technology (CST): GAPDH (#2118, 1:1,000), p-ERK1/2^{Thr202/Tyr204} (#4370, 1:1,000), T-ERK1/2 (# 4695, 1:1,000), p-p38^{Thr180/Tyr182} (#4511, 1:1,000), T-p38 (#9212, 1:1,000), p-JNK^{Thr183/Tyr185} (#4668, 1:500), T-JNK (#9258, 1:1,000), RIP2 (#4142, 1:1,000), p-TAK1^Thr184/187 (#4508, 1:500), T-TAK1 (#5206, 1:500), TGF-β, p-smad3^ser423/425 (#8769, 1:500), and T-smad3 (#9513, 1:1,000).

The gastric tissue micro‐array (TMA) was purchased from Shanghai Outdo Biotech Co. HRP‐conjugated secondary antibody and DAB kit was from ZSbio. Phenylmethylsulfonyl fluoride (PMSF), pepstatin A, metformin and berberine were from Sigma‐Aldrich. Aprotinin and Leupeptin were from GLPBio. EDTA and EGTA were from Solarbio. Human recombinant TGF‐β1 and monoclonal primary antibodies against β‐actin, p‐AMPKα Thr172, AMPKα, p‐Smad3 Ser423/425 and Smad3 were from Cell Signalling Technology. Monoclonal antibody against TGF‐β1 was from Abcam, and monoclonal antibody against LKB1 from Santa Cruz Biotechnology. Lipofectamine3000 was from Thermo Fisher Scientific Inc. FITC‐conjugated donkey anti‐rabbit antibody was from Life Technologies. RNAsimple Total RNA kit was from TIANGENE BioTec. Dual luciferase assay kit was from Promega Corporation. Restriction enzymes XhoI and HindIII and T4 ligase were from New England Biolabs. TGF‐β1 ELISA kit was purchased from Merck.