Collagen 4

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.

Cultured podocytes and kidneys were homogenized in PRO-PREP^TM protein extraction solution (iNtRON Biotechnology, Seoul, Korea) containing protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). All samples were quantified using the Bradford assay (Bio-Rad, Hercules, CA, USA) with BSA as a standard, and an equal amount of each lysate was examined by SDS-PAGE. The separated proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% nonfat dry milk followed by primary antibody incubation at 4°C overnight. Primary antibodies for xBP1 (Santa Cruz Biotechnology), Bip (Santa Cruz Biotechnology), nephrin (Progen Biotechnik, Heidelberg, Germany), Nox4 (Santa Cruz Biotechnology), TNF-α (Cell Signaling, Danver, MA, USA), Collagen IV (SouthernBiotech, Birmingham, AL, USA) were prepared in 0.1% Tris-buffered saline containing Tween-20 and 1% milk at an appropriate dilution. Subsequently, the membranes were washed with phosphate-buffered saline-Tween solution followed by incubation with horseradish peroxidase-conjugated secondary antibody. The bands were visualized with a ChemiDoc^TM XRS+ (Bio-Rad, Hercules, CA, USA) imaging system using a Luminata Forte enhanced chemiluminescence solution (Millipore).

The ECM microarray was fabricated from single-component ECMs or multi-component mixtures of the following ECM proteins: collagen IV (C, mouse, Southern Biotech; Cat. No. 1250–04S), fibronectin (F, bovine, Sigma; Cat No. F1141), laminin (L, mouse, Life Technologies; Cat No. 23017–015), gelatin (G, bovine, Sigma; Cat No. G1393), heparan sulfate (H, mouse, Sigma; Cat No. H4777), and Matrigel (M, mouse, BD Biosciences; Cat No. 356231) (Fig. 1A). The single factor ECMs were further mixed at equal mass ratios (ie, 1:1, 1:1:1, etc.) to obtain a total of 63 unique ECM combinations, each with 6 replicates on each ECM microarray slide. The final concentration of all ECM combinations was held constant at 0.5 mg/ml. The ECMs were deposited using an OmniGrid Accent Microarrayer (Gene Machines) onto a surface-reactive glass slides (Schott H, Nexterion) for covalent protein conjugation, as described previously^{18 (link)}. The resulting combinatorial ECM microarray consisted of individual microenvironments in the form of circular “islands” that were 0.3 mm in diameter and 0.3 mm apart from neighboring islands. After covalent attachment of the ECMs, the microarray slides were air dried and transferred into vacuum sealed boxes and stored in the dark at 4 °C.