Paxillin

1.5 × 10⁶ (8 × 10⁵) cells were seeded in 10 cm (6 cm) plates. A rubber policeman was used to harvest cells gently. The cells were washed in PBS (Thermo Fisher Scientific, Waltham) and lysed in RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% (v/v) IGEPAL CA-630, 0.5% (w/v) Na-deoxycholate, 0.1% (w/v) SDS) supplemented with protease and phosphatase inhibitor cocktail (Roche, Basel). After SDS-Page and wet blot, protein abundance was evaluated via Odyssey infrared scanner (LICOR). Primary antibodies for Rpl7 (A300-741A, Bethyl, Montgomery), Vinculin (sc-25,336, SantaCruz, Dallas), E-cadherin (#3195S), Vimentin (#5741S), Lck (#2984 T, all Cell Signaling, Danvers), p(Y118)-Paxillin (MAB6164), and Paxillin (MAB4259, both R&D Systems, Minneapolis) and p(Y118)-Paxillin (Supplementary Fig. S4, 44-722G, Thermo Fisher Scientific, Waltham) were used.

Cells on fibers were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X100 solution and blocked in 5% goat serum. Paxillin (Tyr31) staining was done using primary rabbit anti-Paxillin antibodies (Invitrogen) at a dilution of 1:250 and incubated at 4 °C for 1 h. Secondary goat anti-rabbit Alexa Fluor 488 (Invitrogen) antibodies were incubated for 45 min at room temperature in the dark. For double immunostaining, total Paxillin (Host: Mouse, Invitrogen) primary antibodies at a dilution of 1:40 and Phospho-FAK (Tyr397) (Host: Rabbit, Invitrogen) primary antibodies at a dilution of 1:200 were used and incubated for 4 h. Secondary antibodies goat anti-mouse Alexa Fluor 647 and goat anti-rabbit Alexa Fluor 488 were used at dilution of 1:150 and incubated for 1.5 h at room temperature. F-Actin stress fibers were stained using Rhodamine Phalloidin (SantaCruz Biotechnologies). Cell nuclei were stained with 300 nM of DAPI (Invitrogen) for 5 min. The scaffolds were kept in 2 ml antifade imaging solution during imaging. Fluorescent images were taken using an Axio Observer Z1 microscope (Carl Zeiss). Actin live cell imaging was performed as per the manufacturer’s instructions on using the reagent CellLight Actin-RFP, Bacman 2.0 (Invitrogen).

We fixed podocytes that had been cultured on collagen-coated coverslips for 14 days with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, blocked with 1% BSA, and immunolabeled with FITC-phalloidin (Sigma-Aldrich, St. Louis, MO, USA), ZO-1 (Invitrogen, Carlsbad, CA, USA) and paxillin (Invitrogen), as well as FOXO3a (Cell Signaling Technology, Danvers, MA, USA). Subsequently, we incubated the cells with Alexa 594-conjugated anti-rabbit antibody (Invitrogen) for 2 hours at RT. The images were collected using an LSM 510 META laser-scanning confocal microscope (Carl Zeiss Microimaging, Thornwood, NY, USA) at the Soonchunhyang Biomedical Research Core Facility of Korea Basic Science Institute (KBSI).

Cultured podocytes grown on collagen-coated coverslips for 14 days were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, blocked with 1% BSA, and immunolabeled with FITC-phalloidin (Sigma-Aldrich) and Paxillin (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. The images were collected using an LSM 510 META laser-scanning confocal microscope (Carl Zeiss Microimaging, Thornwood, NY, USA).

Cells were rinsed with PBS and scraped in ice-cold RIPA lysis buffer (Boston BioProducts) and centrifuged at 8,000 rpm for 5 min at 4 °C. A Coomassie Bradford protein assay (Thermo Fisher) was performed and equal amounts of protein were combined with 4x reducing Laemmli sample buffer (Boston Bioproducts) prior to loading on to SDS-page gel. 20 μg of protein was loaded for each sample and separated by SDS–PAGE, and immunoblotted with antibodies to DRP1 (Novus Biologicals), TRAP1, MFN2, MCT4, VDAC1 (Santa Cruz Biotechnology) NF2, phospho-NF2 (Cell Signaling Technology), FAK (Millipore), Paxillin (Invitrogen) Actin (Sigma). After incubation with peroxidase-conjugated secondary antibodies, the signals were visualized by Clarity™ enhanced chemiluminescence (Bio-Rad) according to the manufacturer’s instruction. Protein band intensity was quantified by ImageJ software.