Rabbit anti p jnk

To determine APN, LC3B, JNK/P-JNK, Akt/P-Akt, P62, and cleaved-caspase-3/pro-caspase-3 levels, proteins were extracted from the cells by suspension in radioimmunoprecipitation assay buffer. Samples were centrifuged at 12,000 rpm at 4°C for 30 min, and the supernatants were recovered for analysis. The protein concentrations were determined using the Bradford protein method and the bicinchoninic acid protein assay kit (Sigma, St Louis, MO, USA). Protein (40 µg) was electrophoresed on a pre-cast bis-Tris polyacrylamide gel (8%~12%) and then transferred onto a polyvinylidene difluoride membrane. Membranes were blotted with rabbit anti-APN (1:1,000), rabbit anti-LC3B (1:500), rabbit anti-JNK (1:1,000), rabbit anti-p-JNK (1:1,000), rabbit anti-Akt (1:1,000), rabbit anti-p-Akt (1:1,000), mouse anti-P62 (1:1,000), rabbit anti-Caspase-3 (1:1,000) (all from Abcam, San Francisco, CA, USA), and mouse anti-actin (1:1,000; Proteintech, NY, USA), followed by horseradish peroxidase-conjugated secondary antibodies (1:5,000; ZsBio, Beijing, China). Immunoblots were visualized using enhanced chemiluminescence (LAS-4000).

The frozen kidney tissue mass was taken from the freezer at −80°C, weighed, and fully ground using liquid nitrogen. Lysis buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to extract kidney tissue proteins of rat. Concentration of protein was tested using Coomassie Brilliant Blue. We added a 20-μg protein sample to the SDS-PAGE gel and electrophoresed and transferred it to the PVDF membrane (GE Healthcare Life Scences, China). The membranes were blocked with 5% milk (dissolved in 1% TBS-T solution) for 2 h, and then incubated overnight at 4°C with the mouse anti-GAPDH (1: 2500; Abcam Inc, Cambridge, UK), rabbit anti-bcl-2 (1: 1000; Abcam, Inc, Cambridge, UK), rabbit anti-Bax (1: 2000; Abcam Inc, Cambridge, UK), rabbit anti-p-JNK (1: 4000; Abcam Inc, Cambridge, UK), and rabbit anti-p-ERK1/2 (1: 4000; Abcam, Inc., Cambridge, UK) antibodies. After incubation and washing, membranes were incubated with corresponding secondary antibodies, anti-mouse/rabbit IgG (1: 5000; Cell Signaling Technology, Inc.) that bind to horseradish peroxidase (HRP). According to the chemiluminescence image development, Image Lab software (Bio-Rad, Shanghai China) was used to analyze the optical density values of the strip on the film, and to compare the difference of the relative optical density among the various groups after correction.