Anti immunoglobulin g igg antibody

The Magna RIP kit (Millipore, Bedford, MA, USA) was adopted for RIP assay according to the manufacturer’s instructions. Saos2 and MG63 cells were lysed by RIP lysis buffer (Beyotime, Shanghai, China) as previously described [30 (link)]. The whole cell extract (100 μL) and radio-immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) were incubated together. The magnetic beads pre-coated with anti-argonaute 2 protein (Ago2) antibody (Abcam, Shanghai, China) were added to interact with circ_0078767-miR-889 complex, and anti-immunoglobulin G (IgG) antibody (Millipore, Bedford, MA, USA) served as the control. After the immunoprecipitate and proteinase K were incubated to remove the protein, qRT-PCR was utilized to determine the relative enrichment of circ_0078767 in the immunoprecipitate.

Cells of A172 and U87 were fixed for 10 minutes to generate DNA‐protein cross‐links. After shearing into 200‐ to 1000‐bp chromatin fragments, cell lysates were immunoprecipitated with anti‐MYC or normal control anti‐immunoglobulin G (IgG) antibody (Millipore). The retrieved chromatin DNA by beads was subjected to qRT‐PCR analysis.

ZNF687-overexpressing cells (2 × 10⁶) were treated with 1% formaldehyde to cross-link proteins to DNA in a 100-mm culture dish. Cell lysates were collected in a 15-ml tube and sonicated to shear the DNA into 300–1000-bp fragments. Aliquots containing equal amounts of chromatin supernatants were incubated with 1 μg anti-FLAG (Abcam) or anti–immunoglobulin G (IgG) antibody (Millipore) overnight at 4 °C with rotation. Following reverse cross-linking of protein/DNA complexes to free the DNA, real-time PCR was performed.