Identification of Target Protein MHY9 with RhoB

Abstract: To identify the target molecules associated with active RhoB， GST-fusion protein of the active form of RhoB (GST-RhoB) was prepared and immune-precipitated with LPS-treated DC2.4 cell lysates. The precipitated samples were subjected to SDS-PAGE and then was stained with Colloidal Gold Total Protein Stain. Molecular mass analysis of the protein which was associated with GST-RhoB in the lysates was performed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). With dendritic cells (DCs) which were prepared from mice incubating with or without LPS for 12 hours， the subcellular colocalization of RhoB and its interacting partner were detected by confocal microscopy after labeling with antibody. The results indicated that motor molecule MYH9 was identified as a novel associating protein of active RhoB by MALDI-TOF mass spectrometry. Confocal microscopy analysis revealed that MYH9 colocalized with RhoB in DCs regardless of LPS stimulation. These results suggest that MYH9 is associated with RhoB and it may be a specific target of RhoB.

CSTR: 32200.14.cjcb.2013.03.0013