Myh14

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 MYH9 (Proteintech, Rosemont, IL, USA), MYH10 (Proteintech), MYH14 (Proteintech), nephrin (Progen Biotechnik, Heidelberg, Germany), NADPH oxidase 4 (NOX4, Santa Cruz Biotechnology), α-actinin-4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), β1 integrin (Santa Cruz Biotechnology), and synaptopodin (Synaptic Systems, Gottingen, Germany) 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) imaging system using a Luminata Forte enhanced chemiluminescence solution (Millipore).

Podocytes were grown on collagen-coated coverslips for 14 days, fixed with 4% paraformaldehyde (PFA), permeabilized with 0.25% Triton X-100, blocked with 1% BSA, incubated with primary antibodies for 1 h at room temperature, and finally incubated with secondary antibodies for 1 h at room temperature. Primary antibodies against the following proteins were used: ZO-1 (Invitrogen), syanptopodin (Synaptic Systems), MYH9 and MYH10 (a gift from Dr. Lawrence B. Holzman)^{56 (link)}, MYH14 (Proteintech), ZO-1 (Invitrogen), FITC-phalloidin (Sigma-Aldrich), and Rhodamine-phalloidin (Invitrogen). The images were collected using an LSM 510 META laser-scanning confocal microscope (Carl Zeiss Microimaging, Thornwood, NY, USA).