Antibodies used in this study were summarized at the dilutions listed: anti‐METTL14, 1:1000, (IB; Cell Signaling Technology, #: 51104S), anti‐N6‐methyladenosine modifications of RNA and DNA (m6A), 1:2000, (dot blot; Synaptic Systems, #: 202 003); anti‐IRF3, 1:1000, (IB; Cell Signaling Technology, #: 4302S); anti‐pIRF3, 1:1000, (IB; Cell Signaling Technology, #: 4947S) anti‐TBK1, 1:1000, (IB; Cell Signaling Technology, #: 3504S); anti‐pTBK1, 1:1000, (IB; Cell Signaling Technology, #: 5483S); anti‐RIG‐I, 1:1000, (IB; Cell Signaling Technology, #: D14G6); anti‐MDA5, 1:1000, (IB; Cell Signaling Technology, #: D74E4); anti‐FTO, 1:1000 (IB; Cell Signaling Technology, #: D6Z8W); anti‐METTL3, 1:1000 (IB; Cell Signaling Technology, #: E3F2A); anti‐ALKBH5, 1:1000 (IB; Abcam, #: ab195377); anti‐MAVS, 1:500, (IB; Santa Cruz Biotechnology, #: sc‐365333); anti‐FLAG (M2), 1:1000, (IB; Sigma‐Aldrich, #: F1804); anti‐β‐actin, 1:2000, (IB; ZSGB‐BIO, #:TA‐09). CHX (HY‐12320), actinomycin‐D, (HY‐17559), were obtained from MedChemExpress (MCE, NJ, USA); PMA (P1585) and Dynabeads mRNA Purification Kit (#: 61006) were purchased from Invitrogen; Click‐iT nascent RNA capture kit (#: C10365) was purchased from Life Technologies; EpiMark N6‐Methyladenosine Enrichment Kit (E1610S) was purchased from New England Biolabs.
Anti alkbh5
Manufactured by Abcam
Sourced in United States
Anti-ALKBH5 is a protein that is used in research applications to detect and quantify the presence of the ALKBH5 enzyme in biological samples. ALKBH5 is a demethylase enzyme that plays a role in the regulation of RNA methylation.
Automatically generated - may contain errors
Lab products found in correlation
Anti fto, by Abcam (4 mentions)
Anti mettl3, by Abcam (3 mentions)
Anti mettl14, by Abcam (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Anti igf 2 antibody, by Abcam (2 mentions)
Anti rig 1, by Cell Signaling Technology (1 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (1 mentions)
Click it nascent rna capture kit, by Thermo Fisher Scientific (1 mentions)
Anti mda5, by Cell Signaling Technology (1 mentions)
Anti tbk1, by Cell Signaling Technology (1 mentions)
Anti p tbk1, by Cell Signaling Technology (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti p irf3, by Cell Signaling Technology (1 mentions)
Anti irf3, by Cell Signaling Technology (1 mentions)
Anti mettl14, by Cell Signaling Technology (1 mentions)
Anti fto, by Cell Signaling Technology (1 mentions)
Anti mavs, by Santa Cruz Biotechnology (1 mentions)
Epimark n6 methyladenosine enrichment kit, by New England Biolabs (1 mentions)
Anti mettl3, by Cell Signaling Technology (1 mentions)
11 protocols using anti alkbh5
1
RNA N6-methyladenosine Regulation Analysis
2
Bone Marrow Stromal Cell Protein Analysis
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BMSCs were lysed using RIPA buffer (Sigma-Aldrich). Following detecting protein concentration, cell lysate was isolated using 10% SDS-PAGE, followed by being transferred on PVDF membranes. After incubating with primary antibodies (anti-BGLAP, anti-COL1A1, anti-RUNX2, anti-OCN, anti-OPN, anti-METTL14, anti-FTO, anti-ALKBH5, anti-WTAP, anti-RBM15, anti-VIRMA, anti-METTL3, anti-SMAD1, and anti-GAPDH, Abcam, Cambridge) at 4 °C overnight, the membranes were incubated with HRP-conjugated secondary antibody (Abcam) at 25 °C for 1 h. Bands were visualized using immobilon ECL ultra western HRP substrate (Sigma-Aldrich). The original bands are shown in the Supplementary material .
3
Antibody and Protein Sources for VEGFA Research
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Recombinant human VEGFA and VEGFA-neutralizing antibody were purchased from R&D Systems (Minneapolis, MN). Antibodies against METTL3 (#86132), FTO (#31687), AKT (#4685), p-AKT (#4060), and p65 (#8242) (in 1:1000 dilution) were purchased from Cell Signaling Technology (Beverly, MA); anti-ALKBH5 (# ab69325, 1:1000 dilution) was purchased from Abcam (Cambridge, UK); anti-m6A (#202003, 1:1000 dilution) was purchased from Synaptic Systems (Göttingen, Germany); anti-RAC3 (#16117-1, 1:500 dilution) was purchased from Proteintech (Rosemont, IL); anti-p-p65 (#WL02169), anti-MMP-9 (#WL03096), and anti-TWIST1 (#WL00997) (in 1:1000 dilution) were purchased from Wanleibio (Shenyang, China); anti-β-actin (#ab8224, 1:4000 dilution) was purchased from Sigma-Aldrich (St. Louis, MO); and goat anti-rabbit (#ZB-2301, 1:8000 dilution) and anti-mouse IgG (#ZB2305, 1:8000 dilution) were purchased from ZSGB-BIO (Beijing, China).
4
RIP Assay for m6A Binding Proteins
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In accordance with the manufacturer’s instructions, the Magna RIP Kit (17–700, Millipore, MA) was utilized to perform the RIP assay. Specifically, 5 μg of anti-ALKBH5 (Abcam, USA), anti-IGF2BP2 (Abcam, USA), or anti-N6-methyladenosine (m6A) (Abcam, USA) and anti-rabbit IgG (Millipore, Germany) were incubated with 40 μL magnetic beads, prior to the addition of cell lysates (approximately 5 × 107 cells per sample). Next, the RNA–protein IP complexes were washed six times. Following treatment with proteinase K, the RNAs of interest were extracted and purified from the immuno precipitated complex for further qRT-PCR analysis. The relative enrichment was normalized with the input.
5
Investigating BHB and p300 Inhibitor Effects
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The chemical reagents and working concentrations used were as follows: BHB (for cells: 0, 15, and 25 mM; for mice: 100 mg/kg; Sigma-Aldrich, cat. 52,017) and p300 inhibitor A485 (for cells: 2.5 µM; for mice: 30 mg/kg; Sigma-Aldrich, cat. SML2192). The antibodies used were as follows: anti-SLUG (Abcam, cat. ab27568), anti-TWIST1 (Cell Signaling Technology, cat. #90,445), anti-E-cadherin (Abcam, cat. ab231303), anti-vimentin (Sigma-Aldrich, cat. V6630), anti-snail (Sigma-Aldrich, cat. SAB5700806), anti-ZEB1 (Abcam, cat. ab276129), anti-ZEB2 (Abcam, cat. ab191364), anti-GAPDH (Abcam, cat. ab8245), anti-SOX2 (Cell Signaling Technology, cat. #14,962), anti-BMI1 (Abcam, cat. ab269678), anti-CD133 (Abcam, cat. ab284389), anti-KLF4 (Abcam, cat. ab129473), anti-β actin (Cell Signaling Technology, cat. #3700), anti-INMT (Abcam, cat. ab181854), anti-METTL3 (Abcam, cat. ab195352), anti-METTL14 (Abcam, ab220030), anti-ALKBH5 (Abcam, cat. ab195376), anti-FTO (Abcam, cat. ab280081), anti-m6A (Invitrogen, cat. MA5-33030), anti-Ki67 (Abcam, cat. ab15580), and anti-pan BHB-lysine (BHB-K) (PTM BioLabs, China, cat. #PTM-1201RM).
6
Detecting CASC11-UBE2T mRNA Interaction
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For detection of the interaction between CASC11 and UBE2T mRNA, MS2bs (MS2-binding protein)-MS2bp (MS2-binding sequences)-based RIP was performed according to the previous studies (9 (link), 17 (link)). Detailedly, cells were transfected with pLV-MS2, pLV-CASC11-MS2, or pLV-CASC11-mut-MS2, and pMS2-GFP (Addgene). After 48 h, cells were used to perform the RIP assay via a GFP antibody (Roach) and the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instruction. Besides, the RNA pulled down by CASC11 was detected using qRT-PCR or employed for RNA sequencing (BGI Group, Guangdong, China). For identifying the interaction of CASC11 or UBE2T mRNA with ALKBH5, FTO or YTHDF2, cells were used to conduct RIP under the help of the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. Here, it should be mentioned that anti-ALKBH5, anti-FTO, anti-YTHDF2, and negative control IgG antibodies were purchased from Abcam.
7
Antibody Validation for m6A Pathway
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Anti-METTL3 (western blot [WB] 1:1,000; IHC 1:500; ab195352), anti-FTO (WB 1:1,000; IHC 1:500; ab124892), anti-METTL14 (WB 1:1,000; IHC 1:500; ab220030), anti-ALKBH5 (WB 1:1,000; IHC 1:500; ab174124), goat anti-rabbit immunoglobulin (Ig)G (WB 1:2000; ab6721) and goat anti-rabbit IgG-FITC (immunofluorescence [IF] 1:200, ab6717) antibodies were purchased from Abcam, USA. Anti-MAP1LC3-B (WB 1:1,000; IHC 1:100; IF 1:100; A7198), Anti-MAP1LC3-B (WB 1:1,000; IHC 1:100; IF 1:100; A7198), anti-Bcl-2 (WB 1:1,000; IHC 1:100; A), anti-YTHDF1(WB 1:1,000; IHC 1:100; A18126), anti-YTHDF2 (WB 1:1,000; IHC 1:100; A15616), anti-Ki67 (IHC 1:100; A11390), anti-GAPDH (WB 1:1,000; AC027) and anti-β-actin (WB 1:50,000; AC026) antibodies were purchased from Abclonal, China. Anti-m6A (Me-RIP 1:1,000; ABE572) antibody was purchased from Merck Millipore (Massachusetts, USA). Western blot analyses were performed by standard methods described previously.59 (link)
8
Protein Extraction and Western Blot Analysis
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Cell pellets were charged at specific times and washed twice with PBS. Cell extracts were cleavage in RIPA (Beyotime, China). After 30 min of centrifugation at 12,000 × g, the supernatant was quantified using bicinchoninic acid (BCA) method (Beyotime, China). After quantification, sample was boiled at 100℃ with SDS buffer for 5 min. Protein extractions were separated using 10% or 12% SDS/PAGE gels. After incubated with primary and indicated HRP-conjugated secondary antibodies (Thermo Fisher Scientific, USA), membrane signals were exposed by chemiluminescence system (Bio-Rad, USA). Antibodies used were as follows: Anti-ALKBH5 (1:1000, Abcam, USA), Anti-GAPDH (1:10,000, GeneTex, USA), Anti-PKMYT1 (1:1000, Cell Signaling Technology, USA), Anti-IGF2BP3 (1:1000, Proteintech, USA).
9
Western Blot Analysis of ALKBH5 and EZH2
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SDS polyacrylamide gels (10–15%) and PVDF membranes were used to separate and transfer protein (20 μg) from cells, respectively. TBS-Tween buffer (20 mM Tris-HCl, 5% nonfat milk, 150 mM NaCl, and 0.05% Tween-20, pH 7.5) was used to block the membranes for 1 h at 21 °C after blotting. Then, the membranes were incubated with primary antibodies overnight at 4 °C (anti-ALKBH5, 1:200, Abcam; anti-EZH2, 1:200, Abcam; and anti-β-actin, 1:4000, Santa Cruz). Finally, the membranes were incubated with a secondary antibody (1:5000, Santa Cruz Biotechnology). β-Actin was used as a control. The gray values of the protein bands were measured with ImageJ (NIH, Bethesda, MD, USA). The averages of three independent measurements are presented as the final data.
10
Western Blot Analysis of ALKBH5, IGF-2, and GAPDH
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Protein from the specimens were extracted using lysis buffer, and 20 ug of proteins was loaded into 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk, the PVDF membranes were incubated with primary antibody including anti-ALKBH5 (1:1000; Abcam; Cat. no. ab195377), anti-IGF-2 antibody (1:1000; Abcam; Cat. no. ab9574) and GAPDH (1:1000; Abcam; Cat. no.ab9485) at 4℃ overnight. Next, the PVDF membranes were incubated with secondary antibody conjugated-horseradish peroxidase (1:1000; Proteintech; Cat. SA00001-2), and the bands were visualized using ECL detection reagents (Millipore).
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