Fig. 4

circFARP1 directly interacts with CAV1 and inhibits its degradation. A Silver staining for RNA pull-down assay with the specific biotin-labeled circFARP1 probe in CAF lysates. Red arrows indicate the unique differential band precipitated by the circFARP1 probe. B-D Mass spectrometry (B-C) and western blot (D) analysis of proteins in unique differential bands. CAV1 was identified as a candidate protein interacting with circFARP1. E RNA immunoprecipitation (RIP) assays in CAFs using IgG and CAV1 antibodies. The relative enrichment of circFARP1 was calculated by qRT–PCR. F Dual RNA-FISH and immunofluorescence staining assay indicating the colocalization of circFARP1 (red) and CAV1 (green), with nuclear staining with DAPI (blue). Scale bars, 100μm. G-H The mRNA and protein levels of CAV1 in CAFs transfected with sh-NC or sh-circFARP1. n.s., no significant. I Western blot of the expression kinetics of CAV1 in CAFs transfected with empty vector or circFARP1 plasmid and treated with CHX (100 μg/ml) for 0 h, 5 h, 10 h, or 15 h (GAPDH as a control). J western blot of the expression kinetics of CAV1 in CAFs treated with or without MG132 (20 μM) after CHX (100 μg/ml) treatment for 0 h, 5 h, 10 h, and 15 h (GAPDH as a control). K Immunoprecipitation of CAV1 protein in lysates from CAFs with or without circFARP1 silencing, followed by immunoblotting with an anti-ubiquitin antibody. MG132 (20 μM) was added before cell lysis to inhibit CAV1 degradation. L Immunoprecipitation assays demonstrating the interaction of ZNRF1 and CAV1 in CAFs with the indicated treatments. Data are expressed as the mean ± SD. ***p<0.001