m6A-IP and YTHDF2-RIP assays HeLa-tTA cells grown in tetracycline (tet)-free culture medium in 10-cm dish were transfected with 1 μg plasmids of either pBG-PLAC2 or pBG-PLAC2-mut for 24 h. Cytoplasmic RNA was extracted and subjected to m6A-IP57. Thirty micrograms of RNA was us...
Red小鼠/兔启动装试剂盒(Sigma‑Aldrich)的说明书执行。在PLA和IF共 同的实验方法中,细胞用Alexa Fluor偶联二抗处理:包括山羊抗兔IgG HL Alexa Fluor 488(ab150077,Abcam)、山羊抗小鼠IgG HL Alexa Fluor 488(ab150113,Abcam)、山羊抗 小鼠IgG HL Alexa Fluor647(ab1500115,Abcam)和驴抗兔IgG HL Alexa Fluor...
The PLKO.1-puro vector (Sigma) was used to construct shRNAs targeting YTHDF2 or CNOT1. The lenti-CRISPR vector (Addgene) was used to clone the sgRNAs targeting YTHDF2. The sequences of shRNAs and sgRNAs are shown in Supplementary Materials. 2.3. qRT-PCR Briefly, Total RNA was ...
After treatment with RNase A (20 μg/mL) at 37°C for 30 minutes, the cells were incubated with propidium iodide (50 μg/mL; Sigma-Aldrich) for 30 minutes in the dark. The cell cycle was assessed with flow cytometry (FACS Calibur flow cytometer; BD Biosciences, San Jose, CA). The...
Then, for each RIP reaction, 100 μL of supernatant was incubated overnight with the magnetic bead-antibody complex at 4°C. On the second day, the RNA/protein immunocomplex was extensively washed with RIP Wash Buffer (provided in the kit). The cross-linking was reversed by incubation ...
However, the therapeutic efficacy of the combination of OXA and anti-PD1 antibody was mitigated in Ythdf2LKO mice, indicating the critical role of YTHDF2 in determining the efficacy of combination therapy. Furthermore, analysis of peritumoral tissues from patients treated with the combination of ...
(Sigma) for 48 h. A portion of living cells were collected and subjected to DNA extraction using the QuickExtract DNA Extraction Solution 1.0 (Epicenter). The efficiency of indels was detected by T7E1 assay using the T7 endonuclease (NEB). Residual cells were seeded to the 100-mm-dish ...
An equal volume of chilled 20 × SSC buffer (Sigma-Aldrich) was then added before samples were spotted on the nitrocellulose membrane. After UV crosslinking for 10 min, the membrane was washed with 1 x PBST buffer, blocked with 5% non-fat milk and incubated with anti-m6A antibody ...
We confirmed that the Ythdf2 antibody was applicable to IP (Additional file 1: Figure S9). Compared with Ythdf2−/−, Nrp2 mRNA and other candidates were enriched by Ythdf2 protein in the wild type, which was verified by qPCR (Fig. 6c). To examine whether increased gene expression...
Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), but its pathogenic mechanism remains to be explored. The RNA N6-methyladenosine (m6A) reader, YTH (YT521-B homology) domain 2 (YTHDF2), plays a critical role in the HCC progression. However, t...