Horseradish peroxidase conjugated goat anti rabbit or mouse immunoglobulin g

Cells and kidney tissues were washed with PBS and lysed in the M-PER mammalian protein extraction reagent with protease inhibitor cocktail (Thermo Fisher Scientific Inc., San Jose, CA, USA). Proteins were separated with 8–15% SDS-PAGE and then were transferred onto a nitrocellulose membrane (Millipore, Madrid, Spain) by electroblotting. The membrane was blocked for 1 hour at room temperature and then was incubated overnight at 4 °C with anti-Shh, anti-E-cadherin, anti-Smad2, anti-Smo, anti-Gli-1 (1:1000, Santa Cruz biotechnology, Santa Cruz, CA, USA), anti-fibronectin (R&D system Inc. Minneapolis, MN, USA), anti-Bax, anti-Bcl-2, anti-TGF-β₁ (1:1000, Cell Signaling Technology, Beverly, MA, USA), and α-SMA (1:1000 Abcam Inc. Cambridge, MA, USA) primary antibodies. Subsequently, the membranes were stained with horseradish peroxidase-conjugated goat anti-rabbit or mouse immunoglobulin G (1:2,000, Santa Cruz biotechnology, Santa Cruz, CA, USA). The immunoreactive bands were detected by chemiluminescence (enhanced chemiluminescence; BioFX Laboratories Inc., Owings Mills, Maryland, USA). GAPDH (1:2,000, Santa Cruz biotechnology, Santa Cruz, CA, USA) was used as an internal control.

We lysed kidney tissues with protein extraction solution (PRO-PREP™; iNtRON Biotechnology, INC., Sungnam, Korea). After separation with 15% SDS-PAGE and electrotransfer of the proteins to a PVDF membrane(Millipore, Madrid, Spain), we blocked the membrane for 1 h at room temperature. And then the membrane was incubated overnight with antibodies to SP (1:500; Santa Cruz Biotechnology, CA, USA) and β-actin (1:2000; Santa Cruz Biotechnology, CA, USA) at 4°C. The membranes were subsequently stained with horseradish peroxidase-conjugated goat anti-rabbit or mouse immunoglobulin G (1:5,000, Santa Cruz Biotechnology, CA, USA). The immunoreactive bands were detected by chemiluminescence (enhanced chemiluminescence; BioFX Laboratories Inc., MD, USA). β-actin was used as an internal control.

Kidneys were washed with PBS and lysed in an ice-cold lysis buffer containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Proteins were separated with 10% PAGE and electroblotted onto a PVDF membrane (Millipore, Madrid, Spain). The membrane was incubated with primary antibody raised against NLRP3 (1:1000, AdipoGen, San Diego, USA), ASC, caspase-1, IL-1β, MAVS, LC3, parkin, PINK1, HIF-1α, AIF, cytochrome c, Bax, prohibitin, α-SMA (1:1000, Abcam, Cambridge, UK), GAPDH (1:1000, cell signaling MA, USA), β-actin (1:1000, Santa Cruz, CA, USA) and, subsequently, with horseradish peroxidase-conjugated goat anti-rabbit or mouse immunoglobulin G (1:10000, Santa Cruz, CA, USA). The immunoreactive bands were detected by chemiluminescence (ECL, BioFX Laboratories, Inc. MD, USA). β-actin and GAPDH were used as internal controls of tubular cells and renal tissues. Prohibitin and GAPDH was used for the internal control of mitochondria and cysotol, respectively.