Collagen 1

To examine the changes in renal morphology, we stained formalin‐fixed, paraffin‐embedded sections (3 µm) with haematoxylin and eosin or a Masson's trichrome staining kit (ScyTek Laboratories, West Logan, UT) according to the manufacturer's instructions and as previously described.29, 30 Immunohistochemistry was performed in paraffin sections using a microwave‐based antigen retrieval method.31 The primary antibodies used in this study included antibodies against TNF‐α, IL‐1β, TGF‐β1, fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA), F4/80 (AbD Serotec, Kidlington, UK), phospho‐Smad3 (Rockland Immuno‐chemicals, Gilbertsville, PA), α‐SMA (Sigma, St. Louis, MO), collagen I (Southern Biotech, Birmingham, AL) and phospho‐nuclear factor κ light chain enhancer of activated B cells (NF‐κB)/p65 (Abcam, Cambridge, MA). Positive signals were analysed with the quantitative Image Analysis System (Image‐Pro Plus 7.0; Media Cybernetics, Bethesda, MD) as described previously.32, 33

Proteins from kidney tissues and cultured cells were extracted with RIPA lysis buffer, and western blot analysis was performed as described previously [10 (link)]. After blocking the nonspecific binding with 5% BSA (1h, room temperature), membranes were then incubated with the primary antibody against phospho-Smad3 (Cell Signaling Technology), collagen I (Southern Biotech), Smad7, Smurf2, total Smad3 (Santa Cruz Biotechnology), α-SMA, glyceraldehyde 3-phosphate dehydrogenase (Chemicon, Temecula, CA) overnight at 4°C, followed by the IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA). Signals were captured using the LiCor/Odyssey infrared image system (LI-COR Biosciences, Lincoln, NE) and the intensity of each band was quantified and analyzed by using the Image J software (NIH, Bethesda, MD, USA).

Additional antibodies used for cholangiocyte characterization and signal transduction genes included: keratin 19 (K19, 1:10, Developmental Studies Hybridoma Bank, TROMAIII), collagen I (1:200; Southern Biotech, 1310-01), α-smooth muscle actin (mouse α-SMA, Abcam, ab7817). Secondary antibodies: PE (anti-mouse eBioscience, 12-4010-82, anti-rabbit #12-473981), APC (anti-mouse eBioscience, 17-4010-82). Alexa secondary antibodies- Abcam: goat anti-rabbit 647 (ab150083), goat anti-rat 568 (ab175710), goat anti-rabbit 555 (ab150086).

Protein from the kidney cortex or HK-2 cells was extracted and western blot analysis was performed as described previously47 (link). Antibodies used in this study included: collagen I, collagen IV (Southern Biotech), CTGF, Phospho-p70s6k, p70s6k, β-actin (Santa Cruz), CD32b (R&D), Phospho- NF-κB/p65, NF-κB/p65, phospho-Smad3, Smad3, Phospho-mTOR (Ser2448), mTOR, Phospho-ERK1/2 MAPK, ERK1/2 MAPK, Phospho-p38 MAPK, p38 MAPK (Cell Signaling), and LI-COR IRDye 800-labelled secondary antibodies (Rockland Immuno-chemicals). The detection of specific signals was performed by using the Odyssey infrared image system (LI-COR Biosciences, Lincoln, NE, USA) and quantified by Image J software (National Institutes of Health). The ratio for the protein detected was normalized against β-actin and expressed as the mean ± S.E.M.

Protein from renal tissues were extracted with RIPA lysis buffer and analyzed by Western blotting as previously described [28 (link)-30 (link)]. Briefly, after protein was transferred onto a nitrocellulose membrane, the membrane was incubated at 4°C overnight with primary antibodies against phospho-p65 (ser276), phospho-IκBα (ser32) and IκBα (Cell Signaling), p65, phospho-Smad2/3, Smad7 and Smad2/3 (Santa Cruz), collagen I (Southern Biotech), α-SMA (Sigma), GAPDH (Chemicon, Temecula, CA), followed by the LI-COR IRDye 800-labeled secondary antibodies (Rock-land Immunochemicals, Gilbertsville, PA). The signals were detected with an Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA) and quantified with Image J (National Institutes of Health, Bethesda, MD, USA). The ratio for the protein examined was normalized against GAPDH.

Cells grown on sterile slides in 6 well plates were fixed with 3% paraformaldehyde and permeated with 0.1% Triton X-100. Nonspecific binding was blocked with 1% BSA for 30 minutes. The cells were then incubated with primary antibodies against PIK3R1 (OriGene, Rockville, MD), AKT3 (Santa Cruz), phosphorylated-AKT (Cell Signaling, Beverly, MA), α-SMA (Millipore, Billerica, MA) and collagen I (SouthernBiotech, Birmingham, AL) for 1 hour, followed by secondary antibodies for 30 minutes. Nuclei were counterstained by 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Images were captured using confocal microscope (Nikon Eclipse TE2000-S).

Protein from renal tissues were extracted with RIPA lysis buffer and analyzed by Western blotting as previously described [46 (link), 47 (link)]. Briefly, after protein was transferred onto a nitrocellulose membrane, the membrane was incubated at 4°C overnight with primary antibodies against phospho-p65 (Ser276), phospho-IκBα (Ser32) and IκBα (Cell Signaling), p65, phospho-Smad2/3 and Smad2/3 (Santa Cruz), collagen I (Southern Biotech), α-SMA (Sigma), KIM-1 (Abcam, Cambridge, MA), GAPDH (Chemicon, Temecula, CA), followed by the LI-COR IRDye 800-labeled secondary antibodies (Rock-land Immunochemicals, Gilbertsville, PA). The signals were detected with an Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA) and quantified with Image J (National Institutes of Health, Bethesda, MD, USA). The ratio for the protein examined was normalized against GAPDH.