Fig. 4

MRF binds with HNRNPD to regulate MCP1 expression. A Specific protein binding with MRF was identified by silver staining after RNA pull-down. B The interaction of MRF and HNRNPD was verified by western blotting. C The fragments showing binding between MRF and HNRNPD were analyzed using catRAPID. The organization of HNRNPD domains is shown on the left. D The predicted secondary structure of MRF was determined with the RNAfold Webserver (http://rna.tbi.ac.at/) on the basis of the minimum free energy (MFE) and partition function. E The mRNA levels of HNRNPD, MRF and MCP1 in MSCs were measured by qRT–PCR after HNRNPD knockdown with or without MRF overexpression (n = 3). F The HNRNPD and MCP1 protein levels in MSCs were detected by western blotting, and quantitative results were normalized to the result for GAPDH (n = 3). G MCP1 levels in MSC culture supernatants were quantified via ELISA (n = 3). H The number of migrated monocytes induced by MSCs was decreased after HNRNPD knockdown, and MRF overexpression did not restore the level of MCP1 (n = 3). I The number of migrated monocytes induced by MSC culture supernatants was decreased after HNRNPD knockdown, and MRF overexpression did not restore the level of MCP1 (n = 3). The data are presented as the mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant. All experiments were independently repeated three times