Confirmation of changes in level of secreted proteins. (a) The changes in the secreted proteome of mammalian relative of DnaJ (MRJ) long isoform (MRJ [L]) expressor observed by mass spectrometry were confirmed by Western blot analysis. Serum-free medium from equal number of MDA-MB-435 parent (P), vector (V), MRJ(L) expressors (M) or MDA-MB-231 vector (V), and MRJ(L) expressors (M) was probed for presence of osteopontin (SPP1), osteonectin (SPARC), VGF nerve growth factor inducible (VGF), and zinc binding α2-glycoprotein 1 (AZGP1). Equal amount of total protein extract (20 μg) of the same cells was probed for the level of KiSS1 (melanoma metastasis suppressor); simultaneously, the expression of MRJ(L) was confirmed. β-Actin was used to verify equal loading of the lysate. Apparent molecular weights of the proteins detected are given in parenthesis: VGF (90 kDa), SPP1 (62 kDa), AZGP1 (47 kDa), SPARC (40 kDa), nucleophosmin (NPM1; 37 kDa), MRJ(L) (38 kDa), and KiSS1 (15 kDa). (b) Real-time quantitative RT-PCR analysis was performed using RNA from the MDA-MB-435-vector and MDA-MB-435-MRJ(L) expressor. The PCR primers and probes for KiSS1, NPM1, AZGP1, SPARC, SPP1, and VGF and endorse control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used. The reaction was performed for up to 40 cycles in triplicate. The gene expression Δ CT values of mRNAs from each sample were calculated by normalizing with internal control GAPDH. The fold change is represented as 2-ΔΔCT. Real-time quantitative RT-PCR analysis was performed on the experimental mRNAs in triplicate and the experiment was repeated once from an independent passage.