Fig. 5

CircPCNXL2 interacts with STRAP to mediate ERK/MAPK signaling pathway activation. (a) CircPCNXL2 probe and control probe were applied for RNA pull-down assays in RBE cells, followed by silver staining. A specific band, indicated by the arrow, appeared at approximately 40 kD. (b) The typical STRAP peptide was identified in circPCNXL2-enriched proteins based on MS analysis. (c-d) Western blot showed the levels of STRAP were detected in proteins pulled down by circPCNXL2 probe or control probe in HuCCT1 and RBE cells. (e) RIP assays were performed using STRAP or IgG antibodies. (f) The localization of circPCNXL2 and STRAP were detected by FISH assay and immunofluorescence assays, scale bar = 50 μm. (g-h) The protein levels of STRAP were examined by Western blot in circPCNXL2 overexpression or knockdown HuCCT1 and RBE cells. (i-j) The binding of MEK1/2 to STRAP was confirmed by co-IP with STRAP or MEK1/2 antibody. (k-l) Co-IP assay was applied to investigate the interaction between MEK1/2 and STRAP in the HuCCT1 and RBE cells transfected with circPCNXL2 overexpression or circPCNXL2 knockdown. (m-n) Co-IP assay was used to detect the interaction between MEK1/2 and STRAP in the HuCCT1 and RBE cells. (o) The diagrams of Flag-tagged STRAP truncations mutants. (p) RIP assays were used to detect the enrichment of circPCNXL2 in 293T cells transfected with full-length and truncated Flag-tagged constructs mutants. (q) co-IP assay was performed in 293T cells transfected either with indicated vectors to identify the binding domain of STRAP responsible for its interaction with circPCNXL2. *P < 0.05, **P < 0.01, ***P < 0.001