After extracting total proteins with RIPA Lysis Buffer (Beyotime), the same amount of protein was separated by 10% SDS-PAGE gel and then transferred onto PVDF membranes (Invitrogen). The membrane was incubated with anti-IGF1R (1:1,000, ab263907, Abcam), anti-p-AKT (1:25,000, ab81283, Abcam), anti-AKT (1:500, ab8805, Abcam), anti-cyclin D1 (1:200, ab16663, Abcam), anti-GLUT1 (1:2,500, ab14683, Abcam), anti-YTHDC2 (1:1,000, ab220160, Abcam), or anti-β-actin (1:5,000, ab8226, Abcam) followed by hatching with Goat anti-Rabbit (1:50,000, ab205718, Abcam) or Goat-anti-Mouse (1:5,000, ab205719, Abcam). Protein signals were visualized with the BeyoECL Plus Kit (Beyotime).
Ab220160
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab220160 is a mouse monoclonal antibody that targets the Acetyl-CoA Carboxylase (ACC) protein. It is designed for use in Western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab220163, by Abcam (3 mentions)
Magna rip kit, by Merck Group (2 mentions)
Ab220161, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Beyoecl plus kit, by Beyotime (1 mentions)
Ab205719, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab14683, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab81283, by Abcam (1 mentions)
Protein g magnetic beads, by New England Biolabs (1 mentions)
Hiseq instrument, by Illumina (1 mentions)
Deoxyribonuclease 1, by Solarbio (1 mentions)
Zli 9017, by ZSGB-BIO (1 mentions)
Anti gapdh mouse monoclonal antibody, by Proteintech (1 mentions)
Pv 6001, by ZSGB-BIO (1 mentions)
Chx c7698, by Merck Group (1 mentions)
16 protocols using ab220160
1
Western blot analysis of IGF1R pathway
2
METTL16-mediated m6A RNA Immunoprecipitation
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K1 or BCPAP cells stably overexpressing METTL16 or with METTL16 knocked down and their corresponding control cells were crosslinked by UV (260 nm, 130 mJ/cm2) and harvested in cold PBS. RNA immunoprecipitation (RIP) was performed using a Magna RIP Kit (17–700, Millipore). Briefly, harvested cells were lysed (10% for input) and incubated with an anti-YTHDC2 antibody (1:1000; ab220160; Abcam) overnight at 4 °C. After washing, the immunoprecipitated complex was digested with proteinase K. RNA was extracted, detected via qRT‒PCR and normalized to the input. For m6A RNA binding experiments, total RNA from K1 cells stably overexpressing METTL16 or with METTL16 knocked down was treated with deoxyribonuclease I (Solarbio, China). The RNAs were sonicated and precipitated with Protein G Magnetic Beads (S1430S, NEB) bound to a m6A antibody (202,003, SYSY). After proteinase K (10 µg/mL) enzymolysis, RNAs were isolated for qRT‒PCR analysis (the input served as a control). For m6A sequencing, m6A-RNAs were acquired from the aforementioned m6A-RIP assay. The RNA libraries were created via quality inspection and subsequently subjected to analysis on an Illumina HiSeq instrument. The peaks were visualized with IGV software.
3
Comprehensive Protein Analysis of Tumor Samples
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WB analysis was performed using anti-METTL14, anti-MFN2, anti-FIS1, anti-DRP1, anti-BAX, anti-BCL2, and anti-Caspase7/cleaved-Caspase7 (26158-1-AP, 12186-1-AP, 10956-1-AP, 12957-1-AP, 50599-2-Ig, 12789-1-AP, 27155-1-AP; 1:1000; Proteintech, USA); anti-LC3A/B and anti-p62 (306019, 380612; 1:1000; ZenBio, China); anti-PARP and anti-cleaved-PARP (9542, 5625; 1:1000; CST, USA); and anti-YTHDC2 (ab220160, 1:1000, Abcam, UK) antibodies. The loading control was a mouse anti-GAPDH monoclonal antibody (60004-1-Ig; 1:10000; Proteintech).
For IHC analysis, consecutive tumor sections with a thickness of 4 μm were prepared. Then, deparaffinization and antigen retrieval were performed following the manufacturer’s instructions. These sections were incubated with anti-Ki-67, anti-MFN2 (27309-1-AP; 12186-1-AP; Proteintech) and anti-Caspase7 (T40049S; Abmart, China) antibodies. After incubated with secondary antibodies (PV-6001, ZSGB-BIO, China), the sections were visualized with a DAB chromogenic agent (ZLI-9017, ZSGB-BIO) and observed under a microscope.
For IHC analysis, consecutive tumor sections with a thickness of 4 μm were prepared. Then, deparaffinization and antigen retrieval were performed following the manufacturer’s instructions. These sections were incubated with anti-Ki-67, anti-MFN2 (27309-1-AP; 12186-1-AP; Proteintech) and anti-Caspase7 (T40049S; Abmart, China) antibodies. After incubated with secondary antibodies (PV-6001, ZSGB-BIO, China), the sections were visualized with a DAB chromogenic agent (ZLI-9017, ZSGB-BIO) and observed under a microscope.
4
Comprehensive Analysis of m6A Regulators
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Ferrostatin-1 (S7243), necrostatin-1 (S8037), Z-VAD-FMK (S7023), liproxstatin-1 (S7699), sorafenib (S7397), erastin (S7242), RSL3 (S8155), MG-132 (S2619) were bought from Selleck Chemicals. CHX (C7698) and Act-D (129935) were purchased from Sigma-Aldrich. Anti-N6-methyladenosine antibody (ab208577), anti-METTL3 antibody (ab195352), anti-METTL4 (ab107540), anti-METTL14 antibody (ab220030), anti-FTO antibody (ab92821), anti-ALKBH5 antibody (ab195377), anti-YTHDF1 antibody (ab220162), anti-YTHDF2 antibody (ab220163), anti-YTHDF3 antibody (ab220161), anti-YTHDC2 antibody (ab220160), anti-HNRNPA2B1 antibody (ab31645), anti-ATG3 antibody (ab108282), anti-ATG4A antibody (ab223374), anti-ATG5 antibody (ab108327), anti-BECN1 antibody (ab207612), anti-ATG7 antibody (ab133528), anti-ATG9A antibody (ab108338), anti-ATG12 antibody (ab155589), anti-ATG16L1 antibody (ab187671), anti-LC3-I/II antibody (ab128025), anti-P62 antibody (ab109012), anti-NCOA4 antibody (ab86707), anti-FTH1 antibody (ab65080), and anti-beta actin (ab6276) antibody were purchased from Abcam Technology. Anti-WTAP antibody (sc-374280) was bought from Santa Cruz Biotechnology. Anti-Mouse IgG (G-21040) and anti-Rabbit IgG (G-21234) were bought from Thermo Fisher Scientific.
5
YTHDC2 Binding Partners in Testis
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RIP-qPCR analysis with YTHDC2 antibodies was carried out on P10 to P12 testes from WT and Neurog-cre Rbm46 KO. Testis (100 mg) was lysed in 1 ml of lysis buffer [50 mM tris-Cl (pH 8.0), 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM DTT, 1× protease inhibitor, tRNA (50 μg/ml), and 2 mM Vanadyl RC]. After centrifugation, the supernatants were precleared for 1 hour at 4°C with protein A beads. Protein A beads were incubated with 10 μg of anti-YTHDC2 antibody (Abcam, ab220160) at 4°C overnight. For immunoprecipitation, 1 ml of lysates was incubated with 100 μl of antibody-bound beads with rotation for 4 hours at 4°C. The bead complexes were then washed five times with washing buffer [10 mM tris-HCl (pH 8.0), 150 mM NaCl, 0.01% NP-40, 1 mM MgCl2, and 5% glycerol]. The immunoprecipitates and input samples were treated with proteinase K before RNA extraction. For qPCR analysis, immunoprecipitated RNA was reverse-transcribed using PrimeScriptRT Master Mix (TaKaRa) and analyzed using SYBR Green Premix Ex Taq II (RR820A, TaKaRa). A standard 20-μl reaction volume contained forward and reverse primers (200 nM), 2 μl of cDNA, and 10 μl of SYBR Green Mix.
6
Protein-Protein Interaction Assay
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GST-RBM46 was expressed in E. coli BL21, and YTHDC2 protein with N-terminal His tag was produced by in vitro translation using the TNT quick coupled transcription/translation systems (Promega). GST-RBM46 fusion protein or GST alone in the lysates was immobilized on precleared MagneGST particles (Promega) and then incubated with in vitro translated YTHDC2 protein in binding (Promega). After incubation with rotation for 1 hour at room temperature, the MagneGST particle–protein complexes were washed extensively with washing buffer before addition of SDS-PAGE loading buffer. The following antibodies were used for Western blot: anti-GST (ABclonal, AE001) and anti-YTHDC2 (Abcam, ab220160).
7
YTHDC2 Expression Analysis in NSCLC
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The NSCLC tissues were collected, paraffin‐embedded, and cut into 4 μm serial sections. Sections were incubated with anti‐YTHDC2 (1:500 dilution, Abcam, ab220160) at 4°C overnight, followed by incubation with secondary antibodies. The sections were then visualized under a microscope. YTHDC2 staining intensity was scored as follows: 0 (no staining); 1 (weak); 2 (moderate) and 3 (high). Percentage scores were assigned as follows: 1 (1%–25%); 2 (26%–50%); 3 (51%–75%) and 4 (76%–100%). The final IHC score was calculated by multiplying the intensity score with the percentage of positive cells. YTHDC2 status was regarded as low YTHDC2 expression (score < 4) or high expression (score ≥ 4).
8
Optimizing YTHDC2 Antibody Selection for CLIP and Immunoblotting
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Primary antibodies used for CLIP were rabbit anti-YTHDC2 (Abcam ab220160) and rabbit anti-YTHDC2 (Proteintech 27779-1-AP). We also tested the following antibodies and did not include them in our CLIP protocol due to poor performance: rabbit anti-YTHDC2 (Bethyl Laboratories A303-026A), rabbit anti-YTHDC2 (Bethyl Laboratories A303-025A), and rabbit anti-YTHDC2 (Invitrogen PA5-57920). The following antibodies were used for immunoblots: rabbit anti-YTHDC2 (1:5000; Abcam ab220160), rabbit anti-YTHDC2 (1:1000; Abcam ab176846), mouse anti-β-Actin (1:20,000; Abcam ab6276), and mouse anti-Vinculin (1:500; Santa Cruz Biotechnology sc-73614). Primary antibodies used for histology were 1 µg/mL mouse anti-SYCP3 (Santa Cruz Biotechnology sc-74569), 0.2 µg/mL rabbit anti-γH2AX (Abcam ab11174), and 1 µg/mL rabbit anti-pH3 (Millipore 06-570).
9
RIP Assay for YTHDC2 and Ago2 Binding
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According to the previous report [24 (link)], PC-3 and DU145 cells were lysed with RIP buffer and then hatched with magnetic beads coated with anti-YTHDC2 (1:30, ab220160, Abcam), anti-Ago2 (1:50, ab186733, Abcam) or anti-IgG (1:20, ab6789, Abcam) at 4°C overnight based on the instructions of the Magna RIP Kit (Millipore, Billerica, MA, USA). The enrichment of IGF1R or circMID1 was measured using qRT-PCR.
10
Western Blot Analysis of m6A Regulators
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Cell lysates of MSCs and 293T cells were collected using RIPA lysis buffer containing 1% phosphatase inhibitors and protease inhibitors. After centrifugation at 12,000g for 10 minutes, the supernatant was collected, and total protein concentrations were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of proteins were separated via SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes with a pore size of 0.45 μm (Merck Millipore). PVDF membranes were blocked with 5% skim milk and incubated overnight at 4°C with 1:1000 diluted primary antibodies against MCP1 (ab214819, Abcam), METTL3 (ab195352, Abcam), METTL14 (ab220030, Abcam), METTL16 (17676S, Cell Signaling Technology), FTO (ab126605, Abcam), ALKBH5 (ab195377, Abcam), YTHDF2 (ab220163, Abcam), or YTHDC2 (ab220160, Abcam). Then, PVDF membranes were incubated with horseradish peroxidase–conjugated (HRP-conjugated) secondary antibodies (1:3000) for 1 hour at room temperature. The immunoreactivity was detected using the Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and visualized in the UVP Chemstudio image system (Analytik Jena). The mean intensity ratio of spots was quantified by ImageJ software (NIH), and the expression of GAPDH or β-actin was used as the internal control. See complete unedited blots in Supplemental Figure 4 .
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