Polyclonal goat anti rabbit igg hrp

Lungs were thoroughly homogenized in a homogenizer (Bioprep-6, Allsheng, Hangzhou, China) at 3800 rpm for four cycles, and 0.2 s per cycle, in RIPA buffer (50 mM Tris HCl [pH 7.4], 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl) with protease inhibitors (#11836145001, Roche). For 10 mg of tissue, 500 μL of RIPA buffer was used. After 30 min on ice, the samples were centrifuged at 10,000 × g for 20 min at 4°C, and protein concentration in the supernatant was determined using the bicinchoninic acid (BCA) protein assay (#23225, Thermo Fisher Scientific). Equal amounts of protein from each sample were loaded on SDS-polyacrylamide gel electrophoresed, separated, and transferred onto PVDF membranes. The immunoblots were incubated in blocking buffer [5% skim milk in phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBST)] for 60 min at room temperature and probed with anti-citrullinated histone H3 (#ab5103, Abcam) or anti-GAPDH mAb-HRP-DirecT (#M171-7, Medical & Biological Laboratories) overnight at 4°C. Then, the immunoblots were washed three times for 5 min in PBST, incubated with polyclonal goat anti-rabbit IgG-HRP (#P0448, Dako) for 30 min at room temperature in blocking buffer, and washed three times in PBST again. Immunodetection was performed using a SuperSignal™ West Pico PLUS Chemiluminescent Substrate (#34580, Thermo Fisher Scientific).

The primary and conjugated antibodies used in this study are listed in Table S1. Secondary antibodies used for Western blot were goat anti-mouse IgG HRP (BioRad; 1:3000) and polyclonal goat anti-Rabbit IgG HRP (Dako; 1:5000). Secondary antibodies used for immunofluorescence were donkey anti-rabbit IgG Alexa 594 (Invitrogen A21207; 1:400), goat anti-mouse IgG Alexa 568 (Invitrogen A11004; 1:200) and goat anti-guinea pig Alexa 488 (Invitrogen A11073; 1:200). The PP2 and PP3 compounds were purchased from Merck Chemical Ltd. EGF and PDGF-BB were obtained from Sigma-Aldrich. HGF was obtained from R&D Systems and staurosporine was from Tocris Bioscience. The polyclonal rabbit antibodies against the phosphorylated Y1440 and Y1422 sites on β4 are homemade (method described in supplemental materials).