Figure 5.

Nuclear location of Hsf4 (DBD)-EGFP in human ARPE cells in culture and expression of the endogenous HSF4 and the transgene in various tissues. (A) The ARPE cells were permanently transfected individually with two recombinant constructions: the Hsf4 (DBD)-EGFP hybrid and the full length Hsf4 cDNA-EGFP. Cell extracts were fractionated and immunoblotted. Lane 1: Hsf4 (DBD)-EGFP (123 bp of Hsf4 + 720 bp of EGFP produces a ~32 kDa hybrid gene product). Lane 2: Hsf4-EGFP (approximately 86 kDa hybrid protein). Lane 3: native EGFP. Note that Hsf4 (DBD)-EGFP (~32 kDa) is detected in cytoplasmic (Cyto) and nuclear (Nuc) extracts (open red arrowhead), while Hsf4-EGFP (86 kDa) is predominantly seen in the nucleus. Blue: sequences derived from Hsf4. Green: sequences derived from EGFP. (B) Expression of the endogenous Hsf4 and Hsf4 (DBD)-EGFP hybrid protein in 11 tissues of PND02 transgenic and WT mouse. EGFP, as expected is not seen in the WT. Each blot was made from single tissue samples, thus the lane labeled Le (lens) represents protein sample from one single whole lens. The ratio of the hybrid transgene Hsf4 as determined by densitometry is 0.73 (N1/F2) and 0.58 (N7/F2) in the lens; however, these values may be underestimations considering that high concentrations of protein (crystallins) in the lens mask low concentration gene products, such as transcription factors. The N7/F2 blot was overexposed for detection in tissues with lower expression. Two different antibodies (anti-Hsf4 and anti-GFP) were used for the upper and the lower halves of the immunoblots, respectively. The last lane in N7/F2, control (C), is a cell extract made from permanently transfected ARPE cells expressing hybrid protein Hsf4 (DBD)-EGFP (~32 kDa). A Gapdh pattern also is presented for each immunoblot as an additional control. Ret, retina; Eg, eye globe; Lv, liver; Hrt, heart; Lu, lung; Sp, spleen; Kid, kidney; Si, small intestine; Mu, muscle; Br, brain.