Ago2 antibody

HT1080 cells were seeded on a 96-well plate and transfected with 100 ng of the pCMV6-AC-GFP plasmid expressing G3BP1 (NM_005754; OriGene), an SG marker, using Lipofectamine 3000. One micromolar Cy5-ON was delivered by gymnosis ∼2.5 h after transfection, with or without 1 μM As III. Twenty-four hours later, cells were harvested by trypsin digestion and reseeded into 24-well glass-bottomed plates (MatTeck Corporation). The As III treatment was continued for an additional 24 h, after which the cells were stained with Hoechst 33342 (Thermo Scientific Pierce) and fixed with 4% paraformaldehyde at room temperature for 10 min. Cells were washed once and stored in PBS at 4 °C. Z-section images were acquired using a Zeiss confocal microscope.
For the immunofluorescence analysis, HeLa cells were treated with 1 μM ON and 50 nM Cy5-ON, delivered by gymnosis, with or without 1 μM As III for 24 h. Cells were fixed with 4% paraformaldehyde and permeated with 0.5% Triton X-100 before antibody staining. The Ago-2 antibody (ab57113; Abcam) was used at a dilution of 1:100, and the secondary antibody (Alexa Fluor 555 conjugate, no. 4409; Cell Signaling) was used at a 1:500 dilution.

The cells were rinsed with cold phosphate-buffered saline and lysed by a complete RNA lysis buffer with protease inhibitor and RNase inhibitor from an EZ-Magna RIP RNA-binding protein immunoprecipitation kit (Millipore) according to the manufacturer’s protocol. The cell lysates were stored at −80°C before use and supernatant from cell lysates was collected by high-speed centrifugation. The cell lysate was incubated with RIP immunoprecipitation buffer containing magnetic beads conjugated with human anti-Argonaute2 (Ago2) antibody (Abcam, Bristol, UK) and negative control normal mouse immunoglobulin G (IgG) (Sigma-Aldrich) overnight. The beads were rinsed with cold NT2 buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl₂, 0.5% NP-40), followed by incubation with 10 mg/mL proteinase K buffer. The RNA bound to Ago2 antibody was extracted with TRIzol reagent. Then, the concentration and quality of RNA were measured. Furthermore, purified RNA was analyzed by qRT-PCR. RNA levels of CRNDE were presented as fold enrichment in Ago2 relative to IgG immunoprecipitates.

RIP analysis was conducted using EZ-Magna RIP Kit (Millipore). HEC1-A and Ishikawa cells were lysed in RIP lysis buffer, and then cell lysates were incubated with magnetic beads conjugated with Ago2 antibody or IgG antibody (Abcam). Finally, the precipitated RNAs were purified and measured using qRT-PCR.

The proteins were extracted from BC cells by RIPA buffer (Beyotime, Beijing, China) and quantified with a Protein Quantification kit (Millipore, Billerica, MA, USA). The cell extracts were subjected to Western blot to determine the protein expression of SOX11 and Ago2. GAPDH was used as the protein loading control. Equal amounts of protein extracts were loaded on to SDS-PAGE gel and transferred to the PCDF membrane. After transfer, the membrane was blocked with 5% skim milk, followed by incubation with primary antibodies, SOX11 antibody (1:1000; Abcam, Shanghai, China), Ago2 antibody (1:1000; Abcam, Shanghai, China) or GAPDH antibody (1:1000; Santa Cruz, Santa Cruz, CA, USA). Subsequently, the membrane was washed and incubated with goat anti-rabbit IgG H&L (HRP) secondary antibody (1:2000; Abcam, Shanghai, China). ECL Reagent (Cell Signaling Technology, Danvers, MA, USA) was used for visualization and detection.