Fig. 5
MEK1 interacts with cNFIB and is involved in cNFIB-mediated ERK signaling inactivation and tumor suppression. (A) Fluorescence in situ hybridization (FISH) showed cytoplasmic localization of cNFIB. Scale bars, 20 μm. (B) Total lysates from HuCCT1 cells were subjected to perform RNA pull-down and separated by SDS-PAGE, followed by Coomassie brilliant blue staining and mass spectrometry (MS) analysis. The specific band isolated by cNFIB probe was shown as red arrow. The table showed MS results identifying MEK1 as a potential binding protein of cNFIB. (C) and (D) The cNFIB-MEK1 interaction was validated by RNA pull-down (left) and RNA immunoprecipitation (right) assays in HuCCT1 cells (C) and RBE cells (D), respectively. (E) Western blot showed the expression of p-ERK, t-ERK, p-MEK and t-MEK in HuCCT1 cells transfected with MEK siRNA or RBE cells transfected with vectors expressing wild type (wt) or constitutively activated mutant MEK. (F) Western blot indicated the expression of p-ERK, t-ERK, p-MEK and t-MEK in HuCCT1 cells co-transfected with cNFIB siRNA and MEK siRNA. (G) Western blot showing the expression of p-ERK, t-ERK, p-MEK and t-MEK in in RBE cells co-transfected with indicated vectors. (H) The cell proliferation capacity of the indicated cells was detected by CCK8 assays. (I) Transwell assays showed the migration and invasion capacity in the indicated cells. Data were shown as mean ± SD, unpaired Student’s t test, *P < 0.05; **P < 0.01; ***P < 0.001. MEK1(wt) was a plasmid expressing wild-type MEK1. was MEK1S218E/S222D was a plasmid expressing constitutively activated mutant MEK1. Abbreviations: p-ERK, phosphorylation of ERK; t-ERK, total ERK; p-MEK, phosphorylation of MEK; t-MEK, total MEK