Figure 5From: Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancerConfirmation 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.Back to article page