Fig. 2

The D domain of LRRC4 mediates the LRRC4-ERK1/2 binding and anchors ERK1/2 in cytoplasm. a Alignments of the D domain sequences from LRRC4 and other proteins that contain the D domain, including MEKs, phosphatases, and substrates. b Schematic of the full-length LRRC4 protein, the LRRC4-ΔD mutant protein, and the GST-LRRC4-D fusion protein. c Confocal fluorescence microscopy of HEK293 cells co-transfected with different plasmids to assess the effect of D domain deletion on the co-localization of LRRC4 and ERK1/2. The merged image shows that ERK1 or ERK2 underwent nuclear translocation after the D domain of LRRC4 was deleted. d Co-immunoprecipitation showed that mutation of the D domain disrupted the interaction of LRRC4 and ERK1/2. (WCL: whole-cell lysate). e GST pull-down assays showed that the D domain of LRRC4 pulled down ERK1 and ERK2. Western blot and Coomassie blue staining analysis of whole-cell lysates (WCL) showed the expression of the GST fusion protein. (− : IPTG negative; + : IPTG positive)