For qRT-PCR, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA, and cDNA was synthesized using HiScript III RT SuperMix (Vazyme, Nanjing, China). qRT-PCR was performed using a HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme). Primer sequences are listed in Supplementary Table S2 . IB, IHC, and ELISA were performed using conventional protocols. Primary antibodies included: anti-METTL3 (Abcam, Cambridge, UK), anti-MEX3A (Sigma, St. Louis, MO, USA), anti-IGF2BP3 (Abcam), anti-HA (Abcam), anti-GPX4 (Abcam), anti-SLC3A2 (Abclonal, Wuhan, China), anti-acyl-CoA synthetase long chain family member 3 (ACSL3, Abclonal), anti-ferritin heavy chain 1 (FTH1, Abclonal), anti-insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1, Abcam), anti-YTHDF2 (Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cell Signaling Technology, Danvers, MA, USA). Tissue microarray assay (TMA) slides were obtained and measurement of IHC scores was performed as described in our previous study [26 ]. IGF2BP3, SLC3A2, and ACSL3 proteins were determined in tissues using ELISA kits (Yingxin Biotech, Shanghai, China) in accordance with the manufacturer's instructions.
Igf2bp1
Manufactured by Abcam
Sourced in United Kingdom
IGF2BP1 is a protein that binds to and regulates the expression of insulin-like growth factor 2 (IGF2) mRNA. It plays a role in the post-transcriptional regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Hiscript 2 one step qrt pcr sybr green kit, by Vazyme (1 mentions)
Hiscript 3 rt supermix, by Vazyme (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Anti mettl3, by Abcam (1 mentions)
Anti ha, by Abcam (1 mentions)
Anti ythdf2, by Abcam (1 mentions)
Anti igf2bp3, by Abcam (1 mentions)
Anti gpx4, by Abcam (1 mentions)
0.22 mm pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Magna riptm rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Mettl3, by Cell Signaling Technology (1 mentions)
Normal igg, by Merck Group (1 mentions)
Rbm15, by Abcam (1 mentions)
Igf2bp3, by Abcam (1 mentions)
Igf2bp2, by Abcam (1 mentions)
4 protocols using igf2bp1
1
Comprehensive Gene Expression Analysis
2
Immunohistochemical Analysis of Tumor Samples
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The primary tumor was removed and processed with hematoxylin and eosin (HE) staining and immunohistochemical staining based on the instructions of the procedure. The tumor samples of the nude mice were paraffin-embedded, and fixed with 4% paraformaldehyde. Tissue sections were then cut into slices, dewaxed, and hydrated with graded alcohol. By incubation in 3% H2O2, endogenous peroxidase activity was blocked. Microwave thermal induction, as well as 0.01 M citrate buffer (pH 6.0), was used for antigen retrieval. Immunocytochemistry detection was adopted on tissue slices of 4 μm thickness, with primary rabbit antibodies IGF2BP1 (abcam, Cambridge, United Kingdom) and Ki67 (abcam, Cambridge, United Kingdom) added for overnight incubation at 4°C, and then secondary antibody IgG H&L (abcam, Cambridge, United Kingdom) was added. After being stained by diaminobenzidine, samples were separately assessed by two pathologists.
3
Western Blot Analysis of Protein Markers
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Lysis buffer was used to lyse the cells, and BCA protein assay kit (Thermo Fisher Scientific) tested the concentration of the protein. Then, the protein was isolated with 10% SDS-PAGE and moved to 0.22 mm PVDF membrane (Millipore, Billerica, MA, United States) for electrophoresis. Later on, the membrane was blocked with phosphate buffer saline involving 5% bovine serum albumin for 2 h at room temperature. Afterwards, it was incubated with primary antibodies with different dilutions at 4°C overnight. The primary antibodies were IGF2BP1 (abcam, Cambridge, United Kingdom), GAPDH (abcam, Cambridge, United Kingdom), and Ki67 (abcam, Cambridge, United Kingdom). Goat anti-rabbit IgG H&L (HRP) was the secondary antibody. The protein level in each sample was normalized with respect to GAPDH. Each assay involved 3 repeated wells of each sample and all of them were repeated at least three times.
4
RIP Assay for RNA-Binding Proteins
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RIP assay was carried out using the Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore) as the manufacturer’s instructions. Briefly, cell lysates were incubated with magnetic beads coated with normal IgG (Millipore), RBM15 (Abcam), METTL5 (Proteintech), IGF2BP1 (Abcam), IGF2BP2 (Abcam), IGF2BP3 (Abcam), METTL3 (Cell Signaling Technology), WTAP (Abcam), YBX1 (Abcam) or GFP (Abcam). The RNA-protein complexes were washed and purified and then subjected to RNA extraction by TRIzol. The UBA6 mRNA enriched by indicated antibody was determined by qRT-PCR. MS2-RIP was performed as previous study described [22 (link)].
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