Ab214430

Cardiac muscle tissue was harvested and placed in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride. Protein samples were separated using 10% and 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Biotech Well). The membranes were blocked with 5% BSA in TBST for 2 h and incubated overnight at 4 °C with the following primary antibodies: anti-ALKBH5 (ab195377, Abcam), anti-Mettl3 (ab195352, Abcam), anti-Mettl14 (ab252562, Abcam), anti-FTO (ab280081, Abcam), anti-BAX (ab3191, Abcam), anti-BCL-2 (ab196495, Abcam), anti-cleaved caspase3 (ab214430, Abcam), anti-Raf1 (A0223, Abclone), anti-phospho-Raf1-S259 (AP1012, Abclone), anti-p44/42 ERK1/2 (4370S, CST), anti-FLAG (Abcam, ab1162), anti-Rasal3 (NBP2-83439, Novusbio), and anti-β-actin (4970S, CST). The samples were then incubated at room temperature (24 °C) for 1.5 h with horseradish peroxidase-conjugated secondary antibody. Proteins were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) and gel images were captured using ImageQuant LAS 4000 Mini Biomolecular Imager (GE Healthcare, Barrington, IL, USA).

Melanoma A375 cells were harvested and homogenized in lysis buffer containing phenylmethylsulfonyl fluoride (PMSF). The primary antibodies used were p75^NTR (#ab52987; Abcam, Cambridge, UK), NF‐κB p65 (#8242; CST, Danvers, MA, USA), IκBα (#9242; CST, Danvers, MA, USA), Phospho‐IκBα (#2859; CST, Danvers, MA, USA), caspase‐3 (ab214430; Abcam, Cambridge, UK), caspase‐9 (ab202068; Abcam, Cambridge, UK), Bax (ab32503; Abcam, Cambridge, UK), Bcl‐2 (ab32124; Abcam, Cambridge, UK), Lamin B1 (Beyotime, Shanghai, China), inhibitor of apoptosis protein 2 (c‐IAP2; #380798; ZEN BIO, Chengdu, China), bFGF (#381676; ZEN BIO, Chengdu, China) and β‐actin (Sino Biological, Beijing, China). β‐actin or Lamin B1 expression served as an internal control. For immunofluorescence analysis, cells were incubated with anti‐(human NF‐κB p65) IgG (1 : 100). The primary antibody was detected using a Cy3‐labeled IgG (1 : 100; Beyotime, Shanghai, China), and nuclei were stained with 4',6‐diamidino‐2‐phenylindole dihydrochloride (DAPI). Microscopic analysis was performed using a Nikon E600 microscope (Tokyo, Japan).

The extracted protein lysates from ischemic myocardial tissues and cardiomyocytes were used for Western blot analysis as described previously [27 (link)]. The lysates were normalized to equal amounts of protein, and 10 μg protein from cell lysates or 30 μg protein from tissue lysates were separated by 10% or 12% SDS–PAGE, transferred to polyvinylidene difluoride membrane, and then sealed with 5% nonfat milk in TBST for 3 h. Membranes were incubated with the following primary antibodies: p-AMPK (1:2000, #ab133448, Abcam), AMPK (1:1000, #ab80039, Abcam), PGC-1α (1:1000, #ab191838, Abcam), SIRT3 (1:1000, #ab264041, Abcam), SOD2 (1:5000, #ab13533, Abcam), Bax (1:2000, #ab182733, Abcam), B-cell-lymphoma protein 2 (Bcl-2) (1:2000, #ab182858, Abcam), caspase3 (1:2000, #ab184787, Abcam), cleaved-caspase3 (1:5000, #ab214430, Abcam), and β-tubulin (1:20,000, #66240-1-lg, Proteintech) at 4 °C overnight. Proteins were then probed with specific HRP-conjugated secondary antibodies for 30 min at room temperature. Finally, the protein bands were obtained using the ChemiDoc Touch Imaging System (Chemi Doc, Bio-Rad, Hercules, CA, USA), and the relative intensity of the bands was quantified using Image Lab software.

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)].

C₃H₁₀T_1/2 cells provided by the National Collection of Authenticated Cultures, Chinese Academy of Sciences were plated in a 6-well plate at a density of 8 × 10⁴ cells/well for 24 h and were pretreated with 0, 10^− 8, 10^− 7, or 10^− 6M Tanshinone IIA for 24 h. After incubation in 500µM H₂O₂ for another 24 h, the cells were collected, and total proteins were extracted on ice using radioimmunoprecipitation assay lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China). The protein concentration was detected using an enhanced bicinchoninic acid assay kit (P0010, Beyotime Biotechnology, Shanghai, China). Western blot analysis was conducted as previously described [18 ]. The following antibodies were used: anti-cleaved caspase 3 (ab214430), anti-pro caspase 3 (ab32499), anti-superoxide dismutase 1 (SOD 1, ab13498), anti-heme oxygenase-1 (HO-1, ab68477), anti-catalase (CAT, ab16731) from Abcam (Cambridge, UK) and anti-Nrf2 (12721), anti-β-actin (8457), anti-Keap1 (8047), anti-Bcl2 (3498) and anti-Bax (5023) from Cell Signaling Technology (Danvers, Massachusetts, USA). A chemiluminescent horseradish peroxidase substrate kit (WBKLS0500, Millipore, Billerica, Massachusetts, USA) was used for the electrochemiluminescence detection assay.

Protein concentration was determined using BCA kits (Beyotime Biotechnology). Subsequently, 25 μg of protein was electrotransferred to a polyvinylidene fluoride membrane (Millipore). The membrane was incubated overnight at 4° C with primary antibody against SIRT1 (1:2000, ab110304, Abcam), OXPHOS (1:500, ab110413, Abcam), P53 (1:1000, A5761, ABclonal), CASPASE 3 (1:1000, ab214430, Abcam), BCL2 (1:1000, ab182858, Abcam), BAX (1:1000, ab182733, Abcam). This was followed by secondary antibody incubation (1:1000, Bioss) for 1 hour the next day. Antibody binding was detected using the ECL Western Blotting Substrate (Millipore, WBKLS0500).