Anti p smad2 3

For Western blotting, total protein was isolated from mouse quadriceps or adipose tissue by homogenization in RIPA buffer with complete protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche, Pleasanton, CA)[33 (link)]. Protein quantification was determined by the Bradford protein assay, and samples were equally loaded on 10% polyacrylamide gels (NuPage, Grand Island, NY), electrophoresed at 200V, electrotransferred to PVDF membranes, and probed with antibodies to Atrogin-1 (1:500; ECM Biosciences, Versailles, KY), Myostatin (1:200; Abcam, Cambridge, MA), MuRF1, ZAG (1:500; both Santa Cruz Biotechnology, Dallas, TX), p21 (1:200; Santa Cruz), β-actin, Smad2, p-Smad2/3 (1:1000; all Cell Signaling, Danvers, MA), TGF-β (1:1000; Abcam), and Ezrin (1:1000; BD Transduction Laboratories, San Jose, CA). Quantification was performed by measuring integrated density and normalizing to loading controls. For immunohistochemical analysis, paraffin-embedded tissue sections were stained using polyclonal anti-TGF-β (1:100; Abcam, Cambridge, MA), anti-p21 (1:100, Santa Cruz), or anti p-Smad 2/3 (1:50, Santa Cruz) and the corresponding isotype controls[33 (link)].

Rats were anesthetized and decapitated on a given day, and brain tissues from the ipsilateral peri-infarct area were collected and extracted. Anti-ALK5 (1:1000, Abcam), anti-Smad2/3 (1:1000; Santa Cruz) and anti-pSmad2/3 (1:1000; Santa Cruz) were used as primary antibodies. After the secondary antibody reaction, the bands were visualized by electrochemiluminescence (Millipore, Darmstadt, Germany). The positive pixel area was detected by an image analysis software (BIO-RAD Gel Doc 2000, Watertown, MA, USA). Western blot quantification was performed by densitometry and was normalized to GAPDH. All experiments were conducted under the same conditions and repeated three times.

Kidney tissues were crushed in liquid nitrogen. Then, tissue samples or cell pellets were lysed in NP 40 lysis buffer (50 mM Tris-Cl, pH 8.0. 100 mM NaCl, 5 mM MgCl 2, 0.5% (v/v) Nonidet P-40) on ice for 30 min. The protein concentration was determined using a BCA Protein Assay Kit (Bio-Rad, CA, USA) following the manufacturer's procedure. Samples were run on a 10% SDS-polyacrylamide gel, transferred to an nitrocellulose membrane (Millipore) and immunoblotted with anti-collagen I, IV, anti MMP2, anti-a-SMA, anti-FN, anti-β-catenin, anti-axin2, anti-GSK-3β, anti-akt, anti-JNK, anti-c-jun, anti-c-fos and anti-P-Smad2,3 (Santa Cruz Biotechnology, CA) at 1∶1000 and anti-actin (Santa Cruz Biotechnology, CA) at 1∶5000. Rabbit anti-PA200 serum was diluted 1∶500 into phosphate-buffered saline–0.05% Tween (PBST)–1% ovalbumin. Antigen-antibody complexes were visualized with fluorescent labeled secondary antibodies (Sigma-Aldrich) diluted 1∶5,000 into PBST–1% ovalbumin and Fluorescent Western Blot Imaging Systems (Amersham).

Malonic acid (MA), 2′, 7′-dichlorodihydrofluorescein diacetate (DCF-DA), dimethyl sulfoxide, and epigallocatechin-3-gallate (EGCG), as a positive control of ROS induction, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and trypsin-ethylenediaminetetraacetic acid were purchased from Welgene (Gyeongsan, Korea). SYBR green was purchased from GeneAll (Seoul, Korea). All antibodies (anti-heme oxygenase 1 (HO-1), anti-superoxide dismutase 1 (Sod1), anti-nuclear factor-erythroid 2-related factor-2 (Nrf2), anti-p-inhibitor of NF-κB (IκB), anti-IκB, anti-p-p65, anti-p65, anti-p50, anti-Cox-2, anti-IL-6, anti-TNF-α, anti-p-JNK, anti-JNK, anti-p-ERK, anti-ERK, anti-p-p38, anti-p38, anti-p-c-Jun, anti-c-Jun, anti-c-Fos, anti-p-Smad2/3, anti-Smad2/3, anti-collagen type I alpha 1 (Col1a1), anti-Col3a1, and anti-glyceraldehyde 3-phosphate dehydrogenase (Gapdh)) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Tissue sections on glass slides were detected using Duolink^®in situ Red Starter Kit (Sigma-Aldrich, DUO92101). Slides were blocked with Duolink blocking solution in a preheated humidity chamber for 30 min at 37°C and then incubated with anti-pSMAD1/5/9 (CST, 9511), anti-pSMAD2/3 (Santa Cruz, sc11769), and anti-SMAD4 (Invitrogen, MA5-15682) antibodies in the same chamber overnight at 4°C. Samples were incubated with proximity ligation assay (PLA) probe solution (anti-mouse PLA probe Minus and anti-rabbit PLA probe Plus or anti-goat PLA Plus) for 1 h at 37°C and then incubated with a ligation solution for 30 min followed by amplification reaction for 100 min at 37°C. Slides were mounted with a coverslip using a minimal volume of mounting medium containing DAPI.

Immunofluorescence (IF) was conducted as described previously [38 (link)]. Liquid nitrogen cooled isopentane frozen muscle tissue sections were cut 10 µm thick on a cryostat. The sections were fixed in ice cold methanol for 5 min, rinsed 3 × 3 min in PBS, blocked in 5% FBS, 1% Tween-20 in PBS for 15 min, and incubated with primary antibodies overnight at 4 °C. The primary antibodies were anti-HIS (Santa Cruz at 1:50), anti-p-SMAD2/3 (Santa Cruz at 1:50), dystrophin (Dys2, Novocastra Labs at 1:100), or anti- α-smooth muscle actin (Sigma Chemicals at 1:100). After 3 × 15 min washes, the sections were incubated with species-specific secondary, 488 nm wavelength antibodies for 1 h at room temperature. After three more 15 min washes, the slides were mounted with DAPI and coverslips. Background staining controls were conducted on serial sections by eliminating the primary antibody. These sections displayed no immunogenic staining (data not shown). Pictures were acquired on an Axiophot, with identical exposure settings within each stain on all of the tissues.

The inhibitors used in this study were K02288 (Selleckchem, S7359, Houston, TX, USA), Galunisertib/LY2157299 (Selleckchem, S2230), RepSox (Sigma‐Aldrich/Merck, R0158), A‐83‐01 (MedChem Express, HY‐10432, Monmouth Junction, NJ, USA), Erlotinib (EGFRi, Selleckchem, S1023), and human ALK1 inhibitory antibody (R&D Systems, MAB3701). The hepsin inhibitory antibody (Ab25) was a kind gift from Dr. Kirchhofer, Genentech [10 (link)]. ZFH7116 provided by Dr. Janetka [12 (link)]. The antibodies used in this study were Anti‐sheep HRP (Upstate cell signaling solutions, #12‐342), anti‐rabbit HRP (Millipore, AP132P, Burlington, MA, USA), anti‐hepsin (R&D Systems, AF4776), anti‐GAPDH (CST#2118S), anti‐β‐tubulin (Abcam, ab6046, Cambridge, UK), anti‐pEGFR Y1068 (Abcam, ab40815), anti‐EGFR (Abcam, ab32077), anti‐pSmad2/3 (CST#8828), anti‐Smad2/3 (CST#8685), anti‐Smad2 (CST#5339), anti‐pH3 (Santa Cruz, sc‐8656, Dallas, TX, USA), total‐H3 (CST#9715), anti‐V5 (Invitrogen, #R96025), anti‐Ki67 (Abcam, ab15580), anti‐cleaved caspase 3 (CST#9661), anti‐F‐actin (phalloidin staining) (Thermo Fisher Scientific, A22283), anti‐pSmad1/5 (CST#9516) and anti‐ALK‐1 (R&D Systems, AF370).