Rabbit anti mouse gapdh

To examine protein expressions of p-p65 and Myd88 in A549 cells, total protein extraction kit (Biyun tian biotechnology co., LTD., Shanghai, China) was used to extract A549 cell proteins (Feng et al., 2016 (link)). The protein content was determined by Bradford assay. Equal amounts of proteins (30 μg protein/lane) were electrophoresed using 12% sodium dodecyl sulfate polyacrylamide gels in a Tris/HCl buffer system, followed by electrophoretic transfer to a polyvinylidene difluoride microporous membrane (BioRad, Hercules, PA, USA). Subsequently, the membranes were sealed with 5% skim milk for 2 hours at room temperature and incubated with the appropriate primary antibodies overnight at 4°C: rabbit anti-mouse GAPDH, rabbit anti-mouse p-p65 and rabbit anti-mouse MyD88 (all purchased at Cell Signaling Technology, Boston, Massachusetts, USA). All antibodies were diluted 1/1000. Following 5 times washes of 6 min with Tris-buffered saline with Tween-20 (TBST), immunodetection was accomplished using appropriate horseradish peroxidase-linked secondary antibodies (Cell Signaling Technology, Boston, Massachusetts, USA) and enhanced chemiluminescence system. The Image Lab imaging system (Bio-Rad, California, USA) was used to conduct optical density scanning of the bands, determine the integral optical density value of each band, and conduct optical density scanning analysis.

Total proteins from HUVEC and irradiated intestine were prepared using RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) plus 1× Protease Inhibitor Cocktail (Sigma-Aldrich), 1× Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich) and 1× Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich). Hot-denatured proteins were used for the immunoblotting experiment. Primary antibodies included rabbit anti-mouse total Akt (Cell Signaling Technology, MA, USA), rabbit anti-mouse phosphorylated Akt Ser473(Cell Signaling Technology), rabbit anti-mouse Bcl-xL(Cell Signaling Technology), anti-mouse Bax(Cell Signaling Technology), rabbit anti-mouse caspase 3(Cell Signaling Technology), rabbit anti-mouse cleaved caspase 3(Cell Signaling Technology), rabbit anti-mouse SDF1(Cell Signaling Technology) and rabbit anti-mouse GAPDH (Cell Signaling Technology). Gray density of each sample was analyzed by using Image-Pro Plus Software (Media Cybernetics, Rockville, MD, USA).

Total protein concentrations of lung homogenate lysates were analyzed with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). SDS-PAGE was performed using Mini-PROTEAN^® Precast Mini PAGE Gels (Bio-Rad, Hercules, CA). Trans-Blot Turbo Mini 0.2 µM PVDF Transfer Packs (Bio-Rad) were used for transferring of proteins to the PVDF membranes. Membranes were blocked for 3 h at RT and incubated with primary antibodies (rabbit anti-mouse NF-κB(RelA/p65/) (sc-8008; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse phospho (p)-RelA/p65(NF-κB) (Ser276; A1953; Abcam), mouse anti-mouse arginase-1 (ab239731), rabbit anti-mouse CD206 (ab64693) and rabbit anti-mouse GAPDH (14C10; Cell Signaling; 1:500) in blocking buffer. After washing with PBS-Tween, membranes were incubated with secondary antibodies (Alexa Fluor 488-conjugated goat anti rabbit/mouse (Invitrogen, Carlsbad, CA)) for 1 h RT. Imaging of blots was preformed using a ChemiDoc system (Bio-Rad) followed by quantification with densitometry normalized to GAPDH.

Cell extracts was subjected to SDS–PAGE gels and transferred to polyvinylidene fluori (PVDF) membranes. The membrane was blocked with 5% BSA diluted in PBS and then incubated with primary antibody overnight at 4 °C. The blots were then incubated with secondary antibodies labeled with HRP. Signal was detected using a scanner (ChemiDoc Touch Imaging System, USA). The primary and secondary antibodies used were as below: Rabbit anti-mouse S100A4 antibody (Cell Signaling Technology, USA), Rabbit anti-mouse GAPDH (Cell Signaling Technology, USA) and HRP-conjugated IgGs (Cell Signaling Technology, USA).

Approximately 2–3 × 10⁶ cells were seeded in 10-cm dishes and incubated overnight, after which the cells were rinsed with ice-cold Ca²⁺–Mg²⁺-free PBS (D8537, Sigma-Aldrich) and lysed in 500 μl ice-cold lysis buffer [150 mM NaCl, 50 mM Tris–Cl, pH 7.4, 0.5 mM EDTA, 1% Triton X-100; freshly supplemented with 1 mM NaF, 1 mM NaVO₃, 1 mM PMSF, 1% protease cocktail (Sigma-Aldrich, Cat No P8340), 2% phosphatase inhibitors 2 (Sigma-Aldrich, Cat No P5726), and 2% phosphatase inhibitors 3 (Sigma-Aldrich, Cat No P0044)]. Lysates were gently passed three times through a 27G needle, after which they were cleared by centrifugation at 11,000g for 15 min at 4 °C. Supernatants containing soluble proteins were collected, and protein content was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Cat No 23227). Proteins were resolved on a NuPAGE 4–12% Bis–Tris protein gel (Thermo Fisher Scientific, Cat No NP0332) and transferred onto Immobilon-P PVDF membrane (Merck Millipore, Cat No IPVH00010). Immobilized proteins were detected using respective primary antibodies: rabbit anti-mouse estrogen receptor 1α (1:100; Cell Signaling Technology, Cat No 13258), rabbit anti-mouse GAPDH (1:2000; Cell Signaling Technology Cat No 2118); and a secondary antibody [HRP-linked anti-rabbit IgG (1:2000; Cell Signaling Technology Cat No 7074)].

To obtain protein from cells, RAW 264.7 cells were lysed by RIPA cell lysis buffer. SDS sample buffer was used to denature protein and then protein supernatants were heated at 95°C for 10 min. After quantification by the BCA method (Sigma–Aldrich), the proteins were separated by 10% precast gel (GenScript or KeyGen) via SDS–PAGE. The membranes were incubated with blocking buffer (NCM Biotech) after protein were transferred to PVDF membranes. Rabbit anti‐mouse AhR (Proteintech; or ImmunoWay Biotechnology) and rabbit anti‐mouse GAPDH (Cell Signaling Technology) were used. Images were visualized by the Tanon Gel Imaging System (Shanghai Tanon Co. Ltd.) or the Chemidoc MP Imaging System (Bio‐Rad).

Bcl2, total Smas2/3, p-Smad-2/3, and cleaved caspase-3 expression in mouse cardiomyocytes or mouse cardiac fibroblasts were measured by immunoblot analysis. Proteins extracted from cardiomyocytes and fibroblasts were separated on 12% or 10% SDS-PAGE and then transferred to a polyvinylidene fluoride membrane. After blocking with 5% BSA for 2 h, the membrane was incubated with rabbit mAb Bcl-2 (1:1000, 3498S), rabbit anti-mouse total Smad-2 (1:1000, 5339S), rabbit anti-mouse p-Smad-2 (1:1000, 3108S), rabbit anti-mouse total Smad-3 (1:1000, 9523S, (all from Cell Signaling Technology, Inc. Danvers, MA), rabbit anti-mouse p-Smad-3 (1:1000, ab52903, Abcam, Cambridge, MA), rabbit anti-cleaved caspase-3 (1:1000, 9661S), and rabbit anti-mouse GAPDH (1:1000, 2118S, Cell Signaling Technology) at 4°C overnight followed by incubation with a HRP-conjugated secondary antibody (1:3000, G21234, Thermo Fisher Scientific) for 2 h at room temperature. The resulting signals were detected using the Amersham ECL Prime Western Blotting Detection Reagent (RPN2236, Fisher Scientific). GAPDH was used to ensure equal protein loading.