Ponceau red stain

Fibroblasts were cultured as for RNA analysis. After incubation, cells were lysed into SDS sample buffer for SDS-PAGE and cell conditioned media clarified by centrifugation before concentrating secreted proteins using StrataClean resin at 1:10 (Agilent Technologies, Wokingham, UK) [54 (link)]. For murine fibrosis studies, right lungs were snap frozen and then homogenized in normal saline containing protease inhibitors (Roche, Burgess Hill, UK) before solubilization in SDS sample buffer for SDS-PAGE.
Western blotting of cellular or lung lysates was performed for acetyl histone H3, 1:20000 (Merck Millipore, Watford, UK), and of cell-conditioned medium for secreted pro-LOX and LOX, (1:20, Sigma-Aldrich, Poole, UK) using enhanced chemiluminescence detection (ECL+, GE Healthcare, Buckinghamshire, UK). In the case of acetyl H3, membranes were stripped and reprobed using pan histone H3 antibodies (Merck Millipore, Watford, UK); for LOX quantitation, image analysis of Ponceau Red stain (Sigma-Aldrich, Poole, UK) was used a loading control. Densitometry was used for semi-quantitative analysis of the data.

Total proteins (40 μg per sample) were resolved by one-dimensional SDS-PAGE in quadruple replicates. Proteins were transferred onto nitrocellulose membrane and stained with Ponceau Red stain (Sigma, USA) for visualizing protein loading control. Membranes were de-stained, washed in TTBS buffer (500 mM NaCl, 80 mM Tris.HCl, pH 7.6, 0.1 % Tween 20) for 1 h and incubated in TTBS buffer supplemented with 25 μg/ml concavalin A for 1 h. Membranes were then washed for 30 min in TTBS and incubated for 30 min in TTBS with 50 μg/ml horseradish peroxidase before being washed again in TTBS for 45 min. N-glycosylated protein bands were visualized with detection buffer (45 mg 4-chloro-1-naftol, 15 ml methyalcohol, and 60 ml 10 mM Tris-HCl pH 6.8) by drop-wise addition of a 25 μl aliquot of hydrogen peroxide (H₂O₂, up to 100 μl) until membrane saturation.

Cell lysates were prepared on ice with RIPA buffer (R0278; Sigma-Aldrich) substituted with protease and phosphatase inhibitors (1861284; Thermo Fisher Scientific). Proteins were separated by 4–12% gradient SDS-PAGE in Tris-HCl buffer and transferred to nitrocellulose membranes. Equal transfer was verified with Ponceau Red stain (P7170-1L; Sigma-Aldrich). Membranes were blocked in 5% nonfat milk (42590.10; Serva) and probed with primary antibodies overnight at 4°C. Anti-P21 (2947S) was obtained from Cell Signaling Technology. Anti-CD47 (AF4670) antibody was purchased from R&D Systems. Anti-CD22 (ab218340) and anti-CD24 (ab179821) were obtained from Abcam. As a loading control, anti-GAPDH (2118) antibody was used from Cell Signaling Technology. Membranes were washed and goat anti-rabbit IgG-horseradish peroxidase (1:2,500; RPN4301; Amersham), goat anti-mouse IgG-horseradish peroxidase (1:1,500; A8924; Sigma-Aldrich) or donkey anti-sheep IgG-horseradish peroxidase (1:4,000; A3415; Sigma-Aldrich) were used as secondary antibody. Membranes were developed using chemiluminescence reagents (NEL104001EA; Perkin Elmer) and the X-Stella system (Reytest Isotope Messgeraete GmbH). Image analysis was performed using the AIDA Imaging analyzer (Reytest Isotope Messgeraete GmbH).