Orb178392

Kidney tissues were ground and homogenized and centrifuged at 10,000 rpm about 10-30 min. The supernatant was taken for the next experiments. The protein concentration was measured by the BCA kit (Solarbio, Beijing, China). 40 μg of the protein sample was mixed with 10% SDS gel buffer with a ratio of 1 : 1. The protein was denatured by heating at 95°C for 5 min. The PVDF membrane (Merck, Darmstadt, Germany) was rotated at 80 V for 30 min. It was then blocked with 5% skim milk powder in TBST for 1 h at 4°C. Next, TBST solution containing 3% bovine serum albumin was used to dilute HSP70 (1 : 500, orb228105), P65 (1 : 500, orb229138), p-P65 (1 : 500, orb304662), IκBα (1 : 500, orb223182), p-IκBα (1 : 500, orb223035), and β-actin (1 : 2000, orb178392) (all Biorbyt Ltd., Cambridge, UK). The reaction was remained overnight at 4°C. Prior to testing, the antibody was rewarmed and then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1 : 1000, ABIN101988, antibodies-online, Aachen, Germany) for 1 h. It was washed with ECL luminescent substrate for 3-5 min. The expression levels of proteins were normalized by β-actin.

Total protein was extracted from both tissues and cells using a total protein extraction kit (cat. no. BC3640-50T; Beijing Solarbio Science & Technology Co., Ltd). Protein concentration was measured using a BCA kit (Solarbio, Beijing, China). A total of 40 μg of each protein sample was mixed with 10% SDS gel buffer at a ratio of 1:1 and the protein denatured by heating at 95°C for 5 min. The PVDF membrane (Merck, Darmstadt, Germany) was rotated at 80 V for 30 min, and then blocked with 5% skimmed milk powder in TBST for 1 h at 4°C. Next, the membranes were incubated overnight at 4°C with rabbit anti-rat NLRP3 (1:500, DF7438, Affinity Biosciences), OX42 (1:500, orb176288), and p-JNK1 (1:500, orb312293); polyclonal rabbit anti-rat MAP1LC3A (1:500, orb378164), MAP1LC3B (1:500, orb382715), beclin1 (1:500, orb227780), PI3KC3 (1:500, orb382585), caspase-1 (1:200, orb213639), and β-actin (1:2000, orb178392) (all from Biorbyt) antibodies diluted in a TBST solution containing 3% BSA. Before testing, the membranes were rewarmed, and then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:1000, ABIN101988, antibodies-online) for 1 h. Then, the membranes were washed with an ECL substrate for 3–5 min. Protein expression levels were normalized to those of β-actin, measured through grayscale scanning, and quantified using ImageJ.

The concentration of protein in the sample was measured using a BCA Protein Quantification Kit (Solarbio, Beijing, China). Forty micrograms samples were mixed with 10% SDS-PAGE before being transferred to PVDF membranes (Millipore, Massachusetts, USA). The membranes were blocked with 5% degreased milk powder for 1 h. The primary antibody of each protein was diluted with 5% BSA as follows: rabbit anti-rat Bax(1:800, orb224426, Biorbyt, Cambridge, UK), Bcl-2(1:800, orb228150, Biorbyt), caspase-3(1:800, orb10231, Biorbyt), PIK3CA (1:800, orb228203, Biorbyt), PI3K (1:800, orb137259, Biorbyt), p-PI3K (1:800, orb338965, Biorbyt), Akt (1:800, orb213545, Biorbyt), p-Akt (1:800, orb222951, Biorbyt), and β-actin (1:2000, orb178392, Biorbyt). All primary antibodies were reacted overnight at 4 °C, then incubated with the secondary goat anti-rabbit Ig G (1:1500, ab6721, Abcam, Beijing, China) for 1 h.