Figure 1

Identification of a novel plasma membrane protein, obif, and its subcellular localization. (A) The strategy of microarray-based screening to identify up-regulated genes in chondrocyte differentiations of ATDC5 cell line. (B) Fold changes of representative chondrocyte markers (black) and obif (red) transcripts which are up-regulated in the microarray analysis. (C) Schematic diagram of obif protein structure. The percent sequence identities of each region are shown: SP, signal peptide; TM, transmembrane domain; E-rich, glutamic acid-rich domain. Numbers represent amino acid residues. (D) Detection of FLAG-tagged obif protein. Whole cell lysates of MC3T3-obif, MC3T3-cont, ATDC5-obif, ATDC5-cont, and NIH3T3-obif were electrophoresed by SDS-PAGE. Immunoblots were probed with anti-FLAG M2 antibody or anti-obif antibody. Arrows represent the molecular size of obif protein predicted from its amino acid sequences (29.4 kDa). (E-H) Confocal analysis of overexpressed mouse and human obif in MC3T3-E1 cells. Cells expressing FLAG-tagged mouse obif were stained with anti-obif antibody (red) (E, F). Cells expressing FLAG-tagged human OBIF were stained with anti-FLAG M2 antibody (red) (G, H). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (I) Subcellular localization of obif protein. Subcellular fractions of ST2 cells expressing exogenous mouse obif were analyzed by Western blotting. Immunoblots were probed with an anti-obif antibody to localize obif (top) and with an antibody against β-actin, an abundant cytoplasmic protein (bottom).