Figure 1. Synthesis and secretion of SCRG1 are downregulated in hMSCs after osteogenic commitment.

(a) hMSCs were cultured in osteogenic differentiation medium for 0, 3, 7, 14, and 21-days. qRT-PCR was performed with specific oligonucleotide primers for SCRG1. Transcript expression of SCRG1 was normalized to GAPDH and results are indicated as fold-decrease relative to the control (day 0). Data are presented as mean ± SD. *p < 0.05 was considered significant. (b) To examine the subcellular localization of SCRG1, pSCRG1-FLAG was transfected into HEK293 cells. After 48 h, the cells and conditioned medium were collected. The cells were fractionated using the ProteoExtract Subcellular Proteome Extraction Kit. Five-fold concentrated conditioned medium (CM), cytosol (Cso), membrane/organelle (M/O), nucleus (Nuc), and cytoskeleton (Csk) fractions were analyzed by western blotting with anti-SCRG1 or anti-FLAG antibody. (c) hMSC cultured medium was applied to a Superdex 75 pg column equilibrated with 10 mM Tris-HCl, pH 7.5, containing 0.5 M KCl and chromatographed at 1.0 mL/min with the same buffer. Collected fractions were analyzed by western blotting with anti-SCRG1 antibody (upper panel). Chymotrypsin and ribonuclease were used as molecular mass standards (lower panel). Although cropped blot/gel were used, the gels were run under the same experimental conditions.