Fig. 3

SGO1-AS1 RNA interacts with the PTBP1 protein. a. Identification of SGO1-AS1-associated proteins by an RNA pulldown assay. The proteins pulled down by SGO1-AS1 or the antisense RNA of SGO1-AS1 incubated with SGC7901 cell extracts were resolved by SDS-PAGE and subjected to silver staining. A specific band was identified in the SGO1-AS1 group and is marked with a red box. b. Western blot analysis validation of the biotin-labeled RNA pulldown assay using sense or antisense probes in SGC7901 cells. GAPDH was used as a negative control. c. RIP-qPCR detection of the indicated RNAs retrieved by specific antibodies in MKN28 cells. The fold enrichment of SGO1-AS1 relative to IgG was determined by qRT-PCR. U6 RNA and GAPDH were used as negative controls. d. Serial deletions of SGO1-AS1 were used in the RNA pulldown assays to identify the core regions of SGO1-AS1 required for a physical interaction with PTBP1. Left panel: graphic illustration of the SGO1-AS1 probes. e. RNA pulldown assay was performed using biotin-labeled 1–415 nt (P4) and PTBP1-binding site mutated RNA (P4 mut) of SGO1-AS1, followed by a Western blot analysis. f. RIP assays were performed using an anti-Flag antibody in HEK293T cells transfected with Flag-tagged PTBP1 or its deletion mutants. qRT-PCR was used to measure the enrichment of SGO1-AS1. Error bars represent SD. *P < 0.05, ***P < 0.001