Ep568y

After isolation, content determination and electrophoresis, proteins were electroblotted [31 (link)] and incubated with a monoclonal rabbit anti-phosphorylated SMAD3 (1:1000; S423 + S425, EP823Y; Abcam), anti-SMAD3 (1:1000; EP568Y; Abcam), polyclonal rabbit anti-TGFβ-RI (1:200; Santa Cruz Biotechnology), anti-TGFβ-RII (1:200; Santa Cruz Biotechnology), mouse anti-VDR (1:100, D-6; Santa Cruz Biotechnology), anti-α-smooth muscle actin (α-SMA, 1:000 Dako Cytomatic, Glostrup, Denmark), anti-total tubulin and anti-GAPDH antibodies (Sigma Aldrich) following horseradish peroxidase conjugate goat anti-rabbit or anti-mouse IgGs (Pierce, Rockford, IL, USA). Specific complexes were revealed and quantified and densitometric blot analysis performed in three independent experiments [32 (link)].

Rabbit anti-Smad2 (ab63576), anti-Smad2 around the phosphorylation site of serine 467 (ab63576), Anti-Smad3 (EP568Y), anti-Smad3 phosphorylated on Serine 423 and 425 (ab52903), anti-Smad4 (EP618Y), anti-CD31 (ab28364), anti-CD34 (ab8536) and goat anti-rabbit HRP (IgG H&L) (ab6721) antibodies were purchased from Abcam trading (Shanghai) co., ltd. (Shanghai, China). Anti-CD 146-coated dynabeads were purchased from Invitrogen Corporation (CA, United States).

Sectioned lung tissues were first blocked using 5% w/v bovine serum albumin for 1 h, followed by incubation with the primary antibodies against Smad3 (EP568Y, Abcam), phospho-Smad3 (ab52903; Abcam), TGFβ1 (Ab179695; Abcam), RhoA-GTPase (26904; New East Biosciences), AhR (Ab84833; Abcam), and EpCAM (G8.8; ThermoFisher), respectively, overnight at 4°C. The sample sections were then incubated with secondary antibodies conjugated with Alexa Fluor dyes (ThermoFisher) at room temperature for 1 h. Isotype-matched negative control antibodies (R&D Systems) were used under the same conditions. The nuclei were counterstained with 6-diamidino-2-phenylindole, dihydrochloride (Solarbio, Beijing, China). Sections were mounted with the ProLong Gold Anti-fade Kit (Molecular Probes) and observed with a Nikon Eclipse Ti-U microscope equipped with a DS-Fi2 camera (Nikon). To determine the fluorescence signal in tissue sections, fluorescent-positive cells in four different high-power fields (500 μM²) from each slide were quantified using ImageJ v1.50e (National Institutes of Health). At least two tissue sections per mouse were included for analysis and presented as mean fluorescence intensity per square micrometer.