Luminata forte enhanced chemiluminescence solution

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).

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).

Cultured podocytes were homogenized in PRO-PREP^TM protein extraction solution (iNtRON Biotechnology, Seoul, Korea) containing a 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 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (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 RIPK3 (#AHP1797, rabbit, 1:1000, Bio-Rad) and β-actin (sc-4778, 1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were prepared in 0.1% Tris-buffered saline containing Tween-20 and 1% milk at the appropriate dilution. Subsequently, the membranes were washed with PBS-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 Luminata Forte enhanced chemiluminescence solution (Millipore).