Anti-Argonaute 2 AGO2 Rabbit Monoclonal Antibody

Western blot analysis of AGO2 using anti-AGO2 antibody (M00189).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human Hela whole cell lysates,
Lane 2: human K562 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-AGO2 antigen affinity purified monoclonal antibody (Catalog # M00189) at 1:500 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for AGO2 at approximately 97 kDa. The expected band size for AGO2 is at 97 kDa.

All lanes use the Antibody at 1:2K dilution for 1 hour at room temperature.

MTORT1 serves as a miRNA sponge. (A) Enrichment of miRNA candidates in mitochondria in MCF-7 cells. Candidates of miRNA were predicted using miRDB ( ). Relative expression levels of miRNA were measured by quantitative RT-PCR and compared to that of miR-181c , which was reported as a mitochondrial miRNA. (B) Relative expression levels of miRNA candidates in MTORT1 knockdown cells were measured by quantitative RT-PCR and normalized to 18S rRNA. (C–E) RNA immunoprecipitation analysis of MTORT1 (C) , miR-26a-5p (D) , and miR-1297 (E) using antibody against AGO2 in MCF-7 cells. Relative expression levels of AGO2-enriched non-coding RNA were measured by quantitative RT-PCR and compared to those pulled down by IgG. The results are means ± SDs (n = 3). * P < 0.05.
Index in PubMed under a CC BY license. PMID: 34141617

NDRG1-OT1 serves as a sponge for miR-875-3p . A , B Distribution of NDRG1-OT1 in MDA-MB-231 cells under normoxia ( A ) and hypoxia ( B ). Cytoplasmic and nuclear RNA were fractionated in MDA-MB-231 cells under normoxia ( A ) and hypoxia ( B ). Relative abundance of RNA was normalized to the total amount of RNA and detected by qPCR. GAPDH : cytoplasmic marker; BCAR4 : nuclear marker. C Relative expression levels of six predicted miRNAs in MDA-MB-231 cells overexpressing NDRG1-OT1 . The candidate miRNAs binding NDRG1-OT1 were predicted by miRDB ( ). The expression levels of miRNAs were measured by qPCR and normalized to U6 . D RIP analysis of NDRG1-OT1 using antibody against AGO2 in normoxia. The RIP enrichment of the AGO2-associated lncRNA was measured by qPCR and normalized to 18 S rRNA. The relative fold enrichment was calculated as compared to the IgG group. E Expression levels of miR-875-3p in five breast cancer cell lines under hypoxia and normoxia, detected by qPCR. Loading control: U6 snRNA. The relative expression levels in each cell line were compared with those of the normoxic group, respectively. F Relative expression of NDRG1-OT1 in MDA-MB-231 cells overexpressing miR-875-3p , and followed by treatment of miR-875-3p inhibitor (50 nM). Loading control: GAPDH . ( n = 5). G Schematic representation of firefly luciferase constructs containing the sequence of NDRG1-OT1 (WT) and two mutations of miR-875-3p binding sites (sites 1 and 2). H Luciferase reporter assays of NDRG1-OT1 in HEK293T cells overexpressing miR-875-3p . All data shown are the means ± SDs. * P < 0.05.
Index in PubMed under a CC BY license. PMID: 36127332

Western blot analysis of Argonaute 2 expression in (1) HeLa cell lysate; (2) RAW 264.7 cell lysate; (3) C6 cell lysate.