Ab128873

Clinical tissue samples or cells of each group were collected, and RIPA lysis buffer was added to extract total protein. Appropriate amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the protein was transferred to PVDF membrane, blocked in 5% skim milk for 1 h at 25°C, added with primary antibody, and incubated overnight at 4°C on a shaker.^{17 (link)} Specific primary antibodies are as follows: PROM2 (Abcam, ab74997, 1:1000), CTCF (Abcam, ab128873), β-actin (Abcam, ab8226). After incubation with secondary antibodies, electrochemical luminescence reagent was added without light. Image J software was used to analyze the gray value of the strips.

To detect the enrichment of CTCF in the promoter region of IGF2-AS, the EZ-Magna ChIP TMA kit (Merck Millipore, Billerica, MA) was used for ChIP experiments. The OS cells in the logarithmic growth phase were cross-linked and cultured with 1% formaldehyde for 10 min. The cells were treated with the protease inhibitor and then sonicated until 200-1000 bp chromatin fragments were obtained. In each group, 100 μL of supernatant (DNA fragment) was added to 900 μL ChIP dilution buffer, 20 μL of 50 × PIC, and 60 μL Protein A Agarose/Salmon Sperm DN to invert and mix at 4°C for 1 h. After centrifugation, the supernatant was taken out, and 20 μL was used as input. In the experimental group, the supernatant was mixed with 2 μg of CTCF antibody (ab128873, rabbit, Abcam Inc.). In the NC group, 2 μg of anti-IgG (ab172730, rabbit, Abcam Inc.) was added, and 60 μL of Protein A Agarose/Salmon was added to each tube. Sperm DNA was inverted at 4°C for 2 h. After centrifugation, the precipitate was washed with 1 mL of low-salt buffer, high-salt buffer, LiCl solution, and TE, respectively. Each tube was eluted twice with 250 mL ChIP Wash Buffer. Then, 20 mL of 5 M NaCl was used to reverse cross-link. The DNA was recovered, and the promoter sequence of IGF2-AS in the complex (F: 5′-AGAATTCAGGGGCCCCATCC-3′, R: 5′-CTGCATCTGCACTCAGACGG-3′) was conducted with quantification.