Ab75183

Mice were sacrificed at various time points after bleomycin treatment under anesthesia. The trachea was cannulated, and the left lungs were inflated with 0.5 mL of 10% neutral buffered formalin. Mouse lung tissue was fixed, embedded in paraffin, sectioned to 5 μm slices for Masson’s trichrome13 (link)49 (link) and BMPER staining. Anti-BMPER (ab75183, 1:200, Abcam) and anti-rabbit HRP-DAB cell and tissue staining kit were used according to the manufacturer’s instructions (R&D systems, Minneapolis, MN, USA). The semiquantitative Ashcroft score was used to score pulmonary fibrosis50 (link). In short, upon 200× magnification, each successive field was given a score ranging from 0 (normal) to 8 (total fibrous obliteration of the field). All scores from 5 sections were averaged41 (link).
Paraffin-embedded sections of normal and IPF lungs were used for immunofluorescence staining. Antibodies specific for BMPER (ab75183, 1:200, Abcam) and α-SMA (F3777, 1:250, Sigma, St. Louis, MO, USA) were used for staining. Alexa Fluor-546-conjugated secondary antibody was from Life Technologies. The nucleus were labeled with DAPI (Vector lab, Burlingame, CA, USA), photographed with a Leica TCS SP5 confocal microscope, and analyzed with Leica confocal software.

The expression of BMPER, Collagen1 (Col1), Phospho-Smad1 (p-Smad1), Smad1, Phospho-Smad3 (p-Smad3) and Smad3 were evaluated by western blotting essentially as described33 (link). For western blotting analysis, the following antibodies were used: BMPER (ab75183, 1:1000, Abcam, Cambridge, MA, USA), Collagen1 (ab21285, 1:250, Abcam), p-Smad1 (5753s, 1:1000, Cell Signaling, Danvers, MA, USA), Smad1 (9743s, 1:1000, Cell Signaling), p-Smad3 (9520p, 1:1000, Cell Signaling), Smad3 (9523s, 1:1000, Cell Signaling) and β-actin (12620, 1:1000, Cell Signaling). The secondary antibodies used were Peroxidase AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG (H+L) (Code: 711-036-152, 1:5000, Jackson ImmunoResearch, West Grove, PA, USA) and Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ Subclass 1 Specific, (Code: 115-035-205, 1:5000, Jackson ImmunoResearch,). Pierce ECL Western Blotting Substrate (Cat. # 32106, Life Technologies, Grand Island, NY, USA) was used to visualize on a ChemiDoc MP Imaging System (Bio-RAD, Philadelphia, PA, USA). Quantitative densitometric analysis relative to β-actin was used to aid clarity.