Anti pparδ

Harvested experimental cells were suspended with PRO-PREP (iNtRON Biotechnology, Republic of Korea) and incubated at 4 °C for 1 h. The cell suspension was centrifuged at 13,000 rpm for 30 min at 4 °C to obtain supernatants as protein extracts. Protein samples were separated using 7 or 12% SDS–PAGE. Separated proteins trapped in SDS-gel were transferred to a nitrocellulose membrane. The protein sample-transferred membrane was blocked with 5% skim milk solution, probed with a primary antibody and then reacted with a matched secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The target protein signal was detected using enhanced chemiluminescence (ECL) buffers (Bio-Rad, Hercules, CA, USA). The first antibodies used in this study were as follows: anti-phospho-NFκB (1: 1,000), anti-NFκB (1: 3,000), anti-phospho-IκB (1: 1,000), anti-PPARδ (1: 2,000), anti-SIRT6 (1: 2,000), anti-SOD1 (1: 2,000), anti-nephrin (1: 2,000), anti-caspase 3 (1: 1,000), and anti-β-actin (1: 4,000), which were purchased from Santa Cruz Biotechnology. Anti-cleaved caspase 3 (1:2,000) was purchased from Cell Signaling (Danvers, MA, USA).

The antibodies used were as follows: anti-ICAM-1 (1:1000), anti-VCAM-1 (1:1000), anti-E-selectin (1:1000), anti-phospho NFκB (1:1000), anti-NFκB (1:2500), anti-phospho IκB (1:1500), anti-PPARδ (1:1500), anti-IL-10 (1:1500), and anti-β-actin (1:2000) were purchased from Santa Cruz Biotechnology.