Figure 2

MRJ(L) localizes to nucleus. (a) Comparison of the isoforms of DNAJB6 (mammalian relative of DnaJ [MRJ]). Various domains are indicated with distinct colors. Domains common to both DNAJB6 isoforms bear the same color code. Domains specific to each isoform bear unique color code. The nuclear localization signal (NLS) sequence is represented using single letter code for the amino acids. The amino acids underlined and in bold represent the strongest NLS. (b) COS7 cells were transfected with fusion protein pEGFP-N1-MRJ(L)^WT or pEGFP-N1-MRJ(L)^mut. Cells were visualized 32 hours after transfection using a confocal microscope with 63× water immersion objective. MRJ(L)^WT-EGFP was predominantly in the nucleus, whereas the mutant NLS protein, MRJ(L)^mut-EGFP, exhibits a uniform distribution throughout the cell. (c) Equal protein from nuclear (N) and cytoplasmic (C) extracts (20 μg) from MRJ long isoform (MRJ [L]) expressors of MDA-MB-435 and MDA-MB-231 were analyzed for the presence of MRJ(L). Histone deacetylase 1 (HDAC1) was used as a marker to validate the purity of the nuclear fraction. β-Tubulin was used as cytoplasmic marker. MRJ(L) migrates at the apparent molecular weight of 38 kDa, MRJ short isoform (MRJ [S]) at 28 kDa, and HDAC1 at 55 kDa. EGFP, enhanced green fluorescent protein.