Ab133504

Tissue homogenates and cells were treated with RIPA lysates (Beyotime, China), and supernatants containing proteins were collected by centrifugation. Protein content was assessed by BCA detection kit (EMD Millipore). SDS-PAGE was performed with the same amount of protein samples in each lane, and then the isolated proteins were electrotransferred to PVDF membrane (Millipore). 5% skimmed milk powder was used for blocking the membranes, and then the corresponding primary antibody (eIF5A (1 : 1000, ab32443, Abcam); FANCD2 (1 : 1000, ab108928, Abcam); SLC7A11 (1 : 1000, ab216876, Abcam); HSPB1 (1 : 1000, ab109376, Abcam); Bax (1 : 1000, ab53154, Abcam); Bcl-2 (1 : 1000, ab32124, Abcam); cleaved caspase-3 (1 : 1000, ab32042, Abcam); cytochrome C (1 : 1000, ab133504, Abcam); β-actin (1 : 1000, ab8226, Abcam)) was applied overnight at 4°C. Following that, the membranes were treated for 2 h with the corresponding secondary antibody (Goat Anti-Rabbit IgG H&L (1 : 2000, ab6721, Abcam) and Rabbit Anti-Mouse IgG H&L (1 : 2000, ab6728, Abcam)). The transfer protein on membranes was developed with electrochemiluminescence (ECL, Thermo Fisher Scientific, USA)). Grayscale of the strips was assessed by ImageJ 1.48v software (NIH).

Cells on the slides were rinsed 3 times with PBS, fixed in 4% paraformaldehyde at 4 °C for 15 min, and treated with 0.5% Triton-100 X for 20 min. Then the cell slides were incubated with the primary antibodies against osteocalcin (1:200, ab92552, Abcam) and runt-related gene 2 (RUNX2, 1:100, ab133504, Abcam) at 4 °C overnight. Next, the cells were washed with PBS and incubated with Alexa Fluora or fluorescein isothiocyanate-labeled goat-anti rabbit secondary antibody (1:5000, ab150088, Abcam) at 37 for 1 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole, and the cells were observed under a fluorescence microscope (DM3000, Leica Biosystems, Shanghai, China).

Protein was extracted from the proximal 1 cm portion of the colon tissue using RIPA lysis buffer containing protease inhibitor and quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Then, 20 μg protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocking for 1 h, the membranes were incubated with primary antibodies such as anti-Cytochrome C antibody (ab133504, abcam, Cambridge, UK); anti-Caspase 3 antibody (ab184787, abcam, Cambridge, UK); anti-Cleaved Caspase 3 antibody (ab214430, abcam, Cambridge, UK); anti-NLRP3 antibody (ab270449, abcam, Cambridge, UK); anti-Cleaved-IL-1ββ antibody (mAb#63124, CST, Boston, MA, USA); anti-phospho NF-kB p65 (Ser536) antibody (mAb#3033, CST, Boston, MA, USA); anti-phospho IκBα (Ser32) antibody (mAb#2859, CST, Boston, MA, USA); and anti-GAPDH antibody (AF0006, Beyotime, Shanghai, China) overnight at room temperature. Then, the membranes were incubated with the HRP-conjugated secondary antibody for 1 h. The color was developed with an electrochemiluminescence (ECL) reagent (Beyotime Biotechnology, Shanghai, China) and images were captured using the Bio-Rad gel imager (Bio-Rad, Hercules, CA, USA). The test method was based on previous papers from our research group [26 (link)].

Six and 24 hours after renal I/R injury, the rats were anesthetized, left kidney was harvested. Protein levels of Cyt-C, Caspase-3and MMP-9 were analyzed by Western blot as described previously. [20 ] Briefly, cell lysates were prepared, electrotransferred, and then immunoblotted with anti-Cyt-C (Abcam, ab133504), anti-Caspase-3 (Abcam, ab44976) and anti-MMP-9 (Abcam, ab38898). Detection was performed with Western blotting reagent ECL (Amersham), and chemiluminescence was exposed by the filters of Kodak X-Omat films. After normalizing the bands with the actin control, data analysis was finished by Image Pro Plus (Media Cybernetics, Silver Spring, MD, USA), measuring the densities of immunoreactive bands.