Ab195377

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).

Antibodies against ALKBH5 (Abcam, #ab195377) and YTHDF2 (Abcam, #ab220163) were used in RNA binding protein immunoprecipitation (RIP) assays. The detailed method of RIP assay is described in a previously published paper.²⁷

Cells were harvested and dissolved in RIPA lysis buffer, and the protein concentrations were determined using a bicinchoninic acid (BCA) protein assay (Beyotime Biotechnology, Jiangsu, China). Whole cell lysates were fractionated and transferred to PVDF membranes by an electroblot apparatus. Membranes were incubated at 4 °C overnight with specific primary antibodies. Bands were visualized with an ECL detection reagent (Beyotime). The densitometric quantification was analysed with a β-actin control using Image Lab software (Bio-Rad, Hercules, CA, USA). All antibodies used in this study were obtained from Santa Cruz (CA, USA) and Abcam (Cambridge, UK). The primary antibodies were rabbit monoclonal anti-ALKBH5 (ab195377, Abcam), mouse monoclonal anti-PER1 (sc-398,890, Santa Cruz), mouse monoclonal anti-p-ATM (Ser-1981; sc-47,739, Santa Cruz), rabbit monoclonal anti-p-CHK2 (Thr-68; ab32148, Abcam), rabbit polyclonal anti-p-CDC25C (Ser216; ab47322, Abcam), rabbit monoclonal anti-p-P53 (Ser-15; ab1431, Abcam), mouse monoclonal anti-P21 (sc-71,811, Santa Cruz), mouse monoclonal anti-CYCLIN B1 (ab72, Abcam), rabbit polyclonal anti-p-CDK1 (Tyr15; ab47594), mouse monoclonal anti-CDK1 (A17, Abcam) and rabbit polyclonal anti-β-ACTIN (ab8227, Abcam).

Tissues were homogenized in RIPA lysis buffer containing a 50× protease inhibitor cocktail. Homogenates were then centrifuged at 19,392 g for 15 min to remove cell debris. The supernatant was collected, and total protein concentrations were measured using a protein assay kit (Enhance6d BCA Protein Assay Kit, Beyotime). A volume of 10 μg of the protein was electrophoretically separated in an SDS-PAGE gel (10% Tris–HCl) and transferred to 4.5-μm PVDF membranes. The membranes were blocked with 5% skim milk for 1 h and then incubated with rabbit anti-Mettl3 (1:1,000, ab195352, Abcam), rabbit anti-Mettl14 (1:1,000, ab220031, Abcam), rabbit anti-WTAP (1:1,000, ab195380, Abcam), rabbit anti-β-catenin (1:1,000, #8480, Cell Signaling Technology), rabbit anti-Wnt3 (1:1,000, #2721, Cell Signaling Technology), rabbit anti-S100A4 (1:1,000, ab124805, Abcam), rabbit anti-FTO (1:1,000, ab124892, Abcam), rabbit anti-ALKBH5 (1:1,000, ab195377, Abcam), and mouse anti-GAPDH (1:1,000, ab8245, Abcam) overnight at 4°C room temperature. The membranes were then washed in PBS-T (3 times for 10 min each) and incubated with the anti-rabbit secondary antibody (1:1,000, ab288151, Abcam) or anti-mouse secondary antibody (1:1,000, ab150113, Abcam) at room temperature for 1 h. Bands were visualized by ECL and quantitated using ImageJ software.

Cells were lysed in RIPA buffer (P0013B, Beyotime, China) containing the complete cocktail of protease inhibitors (#11836153001, Roche, Switzerland). Protein concentrations were determined with the BCA protein assay kit (P0011, Beyotime, China). Proteins were separated by 4-20% SDS-PAGE, transferred to PVDF film and blotted with antibodies at 4 °C overnight. Secondary antibodies were pre-labelled at room temperature for 1 h. The PVDF film with the target protein was exposed in the visualizer (AI800, GE, USA). Antibodies were purchased from the following: ALKBH5 (ab195377, 1:1000, Abcam), Caspase-3 (A19654, 1:1000, Abclonal), SQSTM1/p62 (A19700, 1:1000, Abclonal), GAPDH (A19056, 1:10000, Abclonal).

For immunohistochemistry (IHC) analysis, tissue slides were deparaffinized and rehydrated through an alcohol series followed by antigen retrieval with sodium citrate buffer, blocked with 5% normal goat serum (Vector) with 0.1% Triton X-100 and 3% H₂O₂ in PBS for 60 min at room temperature and then incubated with appropriate primary antibodies against ALKBH5(1:1000, ab195377, Abcam), ABCA1(1:200, ab18180, Abcam), and Ki67 (1:1000, ab15580, Abcam) at 4 °C overnight. IHC staining was performed with horseradish peroxidase (HRP) conjugates using DAB detection. Nuclei were counterstained with Hoechst.
For immunocytofluorescence (IF), SKCM cells were first treated with 4% PFA for 15 min. The sections and cells were blocked with Immunol Staining Blocking Buffer (Beyotime) for 1 h and incubated with the primary antibody against ABCA1(1:200, ab18180, Abcam) overnight at 4 °C, then were incubated with Alexa Fluor secondary antibodies (Jackson ImmunoResearch, PA, USA) for 1 h. This was followed by incubation with a diamidinyl phenyl indole (DAPI) coloration for 10 min (Servicebio, China). Images were taken with the fluorescence microscope (IX83, Olympus, Japan).

Cells were fully lysed with ice-cold strong RIPA lysate (CW2333S, CWBIO) on ice for 10 min. Cell lysates were centrifuged at 12,000 g for 10 min at 4°C to remove impurities. The supernatants after centrifugation were the protein solutions, and protein concentrations were quantified by BCA protein assay kit (CW0014S, CWBIO). Equal amounts of proteins from each group were electroblotted and separated by SDS-PAGE and electrotransferred onto PVDF membranes (0.45 μm, IPVH00010; 0.2 μm, ISEQ00010; Millipore). After blocking with 5% skimmed milk, the membranes were incubated overnight at 4°C with primary antibodies including DIRAS1 (ab65139, Abcam), METTL3 (ab195352), METTL14 (ab220030), ALKBH5 (ab195377), FTO (ab126605) and GAPDH (ab8245). Subsequently, the membranes were incubated with the corresponding secondary antibody at room temperature for 2 h. The immunoreactive bands were visualized by enhanced chemiluminescence (RPN2105, Amersham), and the gray values of the bands were read by Image J software.