Fig. 5.

Occupancy of HSP70 and cryab promoters by HSF1 and HSF4 is cell-type specific. Native ARPE cells and NIH3T3 cells were processed for ChIP assay with HSF1 and HSF4-specific antibodies. The left panels show agarose gel electrophoresis of the PCR products. The right panels show schematic representation of the data obtained. a Note that in ARPE cells there is no HSF1 on the cryab promoter either in control (unheated, UH) or heat shocked (H) cells, either at 0 h or at 12 h postheat shock; HSF4 is seen only on the cryab promoter both in heated and unheated cells ( blue ovals). There is no detectable HSF4 on the HSP70 promoter; HSF1 is only seen on the HSP70 promoter in unheated cells, and its enhanced binding is seen in heat-shocked ARPE cells (red pentagons). b In NIH3T3 cells HSF1 is seen on the cryab promoter only in heated cells (red pentagons). HSF1 is also present on the hsp70 promoter in both the heated as well as unheated cells (red pentagon), but it is enhanced in heat shocked cells (red pentagons) just as in ARPE cells in a. Note that there is no HSF4 on either the cryab promoter or the HSP70 promoter in NIH3T3 cells under any condition. The first lane of each gel shows the DNA markers (M, bp base pairs), the last lane is the H₂O control. H heat shocked, UH not heat shocked, In input DNA before immunoprecipitation, NS normal serum. The sizes of amplicons are indicated on the right side of the agarose gels with an arrow pointing to the PCR product. The location of the primers for cryab and HSP70 promoters in ARPE and NIH3T3 cells are schematically depicted. This experiment was repeated twice