RIP assays were performed essentially as described in our previously published study [24 (link), 25 (link)]. In brief, cells were lysed using polysome lysis buffer (5 mM HEPES (pH 7.4), 85 mM KCl, 1 mM DTT, 5 mM PMSF, 0.5% NP40, supplemented with RNase inhibitors (Invitrogen, USA) and PIC (protease inhibitors cocktail, Roche, Switzerland)) on ice for 10 min. After centrifugation, the supernatant was collected with 10% of the lysate serving as “input”. The remainder of the lysate was incubated with 50 μl of protein A/G magnetic beads (Life Technologies, USA) coupled with 2 μg of primary antibodies rotated overnight at 4 °C with IgG antibody as the control. RNA was isolated using TRIzol (Invitrogen, USA) and reverse-transcribed into cDNA for qRT-PCR detection using a Takara SYBR green kit (Takara, Japan). Primary antibodies against YTHDF1 (Abcam, ab220162), YTHDF2 (Abcam, ab220163), YTHDF3 (Abcam, ab220161), and N6-methyladenosine (m6A) (Abcam, ab220161) were used.
Ab220161
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab220161 is a lab equipment product manufactured by Abcam. It is designed for use in scientific research applications. The core function of this product is to [CORE FUNCTION DESCRIPTION].
Automatically generated - may contain errors
Lab products found in correlation
Ab220162, by Abcam (4 mentions)
Ab220163, by Abcam (4 mentions)
Ab246514, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ab220160, by Abcam (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Sybr green kit, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
P stat3 9145, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Ab126605, by Abcam (1 mentions)
Ab134045, by Abcam (1 mentions)
Ab125011, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab205606, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
12 protocols using ab220161
1
RIP-qRT-PCR Assay for m6A Readers
2
Immunoblotting of EMT and m6A Regulators
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Immunoblotting was carried out as previously described [44 (link)]. Antibodies against E-cadherin (3195, 1:1000), Vimentin (5741, 1:1000), p-STAT3 (9145, 1:1000), ZEB1 (70512, 1:1000) and β-actin (4970, 1:1000) were purchased from Cell Signaling Technology. Antibodies against FTO (ab126605, 1:1000), YTHDF1 (ab252563, 1:1000), YTHDF2 (ab246514, 1:1000), YTHDF3 (ab220161, 1:1000), TSG101 (ab125011, 1:1000), and CD81 (ab134045, 1:1000) were purchased from Abcam. Antibodies against IGF2BP1 (22803-1-AP, 1:1000), IGF2BP2 (11601-1-AP, 1:1000), IGF2BP3 (14642-1-AP, 1:1000) and GAPDH (60004-1-Ig, 1:2000) were purchased from Proteintech (China). Full and uncropped blots were uploaded as Supplemental Material.
3
Analyzing YTHDF Proteins in Cardiomyocytes
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Proteins were extracted from heart tissues or isolated mice primary cardiomyocytes with RIPA Lysis Buffer (Beyotime, China), and protein concentration was determined using the BCA protein assay kit (Beyotime, China). Protein samples were separated on a 10% SDS-PAGE gel and then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: YTHDF1 (ab220162, Abcam, USA), YTHDF2 (ab246514, Abcam, USA), YTHDF3 (ab220161, Abcam, USA), ANP (ab262703, Abcam, USA), flag (ab205606, Abcam, USA), and GAPDH (ab8245, Abcam, USA), followed by incubation with secondary antibodies for 1 h. The protein blots were visualized using the ECL kit (Pierce, USA).
4
Protein Expression Analysis by Western Blot
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Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) and the concentration was determined with the BCA Protein Assay Kits (Beyotime, Shanghai, China). A total of 20 µg of protein was loaded for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Samples were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with the following primary antibodies: anti-YTHDF3 (ab220161, Abcam, Cambridge, UK), anti-ZEB1 (21544-1-AP, Proteintech, Wuhan, China), anti-E-cadherin (20874-1-AP, Proteintech, Wuhan, China), anti-N-cadherin (22018-1-AP, Proteintech, Wuhan, China), anti-vimentin (10366-1-AP, Proteintech, Wuhan, China), and anti-GAPDH (ab181602, Abcam, Cambridge, UK). Membranes were then incubated with a secondary horseradish peroxidase-conjugated antibody and visualized with the ECL reagent (Thermo Fisher Scientific, Waltham, USA).
5
Immunohistochemical Analysis of YTHDF3 in TNBC
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IHC staining analysis was performed on paraffin-embedded tissues to assess the protein expression of YTHDF3 in TNBC tissues. Slides were incubated with anti-YTHDF3 (1:800; ab220161, Abcam, Cambridge, UK) and anti-ZEB1 (1:800, 21544-1-AP, Proteintech, Wuhan, China) antibody according to the standard procedure. The IHC staining scores of YTHDF3 were evaluated by 2 independent pathologists blinded to the corresponding patients. The percentage of stained positive cells was scored from 1 to 4 as followed: 1 point for 0–25% of cells stained; 2 points for 26–50% of cells stained; 3 points for 51–75% of cells stained; and 4 points for 75–100% of cells stained. The staining intensity score was calculated from 0 to 3 as follows: 0 points indicates no staining; 1 point indicates weak staining; 2 points indicates moderate staining; and 3 points indicates strong staining. The percentage of positive tumor cells and the staining intensity were multiplied to produce a weighted score for each patient. A score of 8–12 was defined as high expression level and a score of 0–7 was defined as low expression.
6
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.
7
RNA Immunoprecipitation Assay Protocol
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The RIP assay was conducted with Magna RIP Kits (Millipore, Billerica, USA) according to the manufacturer’s instructions. The antibodies used in this study were anti-YTHDF3 (ab220161, Abcam, Cambridge, UK), anti-m6a (202003, Synaptic Systems, Goettingen, Germany), and normal IgG. The RNA complexes were extracted by proteinase K and phenol/chloroform/isoamyl alcohol, and amplified by qRT-PCR.
8
Protein Expression and Western Blot Analysis of Lung Cancer Cells
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Human lung cancer cells were transfected with the relevant plasmids and cultured for 48 h. For western blot analysis, cells were lysed in NP-40 buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1 mM PMSF, and 0.5% NP-40) at 25 °C for 40 min. The lysates were added to 5× loading dye and then separated by electrophoresis. The primary antibodies used in this study were 1:1000 rabbit anti-Flag (sc-166384, Santa Cruz, Dallas, TX, USA) and 1:1000 Abcam (Cambridge, UK) antibody of Myc (ab32072), METTL3 (ab195352), YTHDF3 (ab220161), YAP (ab56701), vimentin (ab45939), E-cadherin (ab1416), cleaved Capase-3 (ab32042), and Tubulin (ab6046).
9
Western Blot Analysis of YTHDF Proteins
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Briefly, B16/F10 cells or tumor tissues were lysed with RIPA buffer (Beyotime Biotechnology, catalog no. P0013B) containing protease inhibitor cocktail (Thermo Fisher Scientific, catalog no. 78425). Nuclear proteins were obtained using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, catalog no. P0027). The protein concentration was evaluated by the BCA Assay Kit (Beyotime Biotechnology, catalog no. P0010S), and 30 μg of total protein were electrophoresed via SDS-PAGE and blotted onto a polyvinylidene fluoride (PDVF, Beyotime Biotechnology, catalog no. FFP33) membrane. After blocking, the PVDF membranes were incubated with various primary antibodies including anti-YTHDF1 (1:2000; Abcam, catalog no. ab220162), anti-YTHDF2 (1:1000; Abcam, catalog no. ab220163), and anti-YTHDF3 (1:1000; Abcam, catalog no. ab220161) at 4 °C overnight. On the next day, the PVDF membrane was incubated with the second antibody (1:5000) for 2 h and washed three times with TBST. The bands were then imaged using a Bio-Rad system following the instructions. The uncropped blots for the main and supplementary figures are provided in the Source Data file.
10
YTHDF3 Immunohistochemistry in Paraffin Sections
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A haematoxylin–eosin (H&E) staining was carried out on paraffin‐embedded sections slides, and IHC staining was performed with an autostainer (link45 System, Dako, Denmark) with anti‐YTHDF3 (ab220161, 1:200 dilution, Abcam). Slides were analysed by Ying Zhang, Jian‐fang Sun and Hao Chen from our hospital.
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