Fig. 6

Absence of a functional REPO domain disrupts the transcriptional kinetics of the hsp70 locus during the heat shock response. a co-IP assays of S2 DRSC cells transiently transfected with plasmids expressing FLAG-tagged Spt5 (FLAG-Spt5) and HA-tagged PhoV164D (HA-PhoV164D). The S2 DRSC cells were subjected to a heat shock and then allowed to recover from the heat shock according to the experimental scheme depicted in Fig. 1a. At the indicated time points in the experimental scheme, cell lysates were prepared and were used for pull-downs using an anti-FLAG antibody. They were later probed by Western blot using an anti-HA antibody. b Quantification of the co-IP assay in a and its comparison with the quantification of the Pho–Spt5 co-IP assay across the same time course. The control lines represent cells that were maintained at 25 °C for the entire duration of the time course detailed in Fig. 1a. Details of the procedure used for quantification can be found in Materials and Methods. c qRT-PCR measurements of nascent hsp70 transcripts produced over the course of the heat shock response from S2 DRSC cells upon over-expression of WT Pho and mutant Pho (PhoV164D), respectively. Distance of location of the primers from the hsp70 TSS is + 687 bp and has been depicted in the form of a cartoon below the data figure. Data information: In b, data are presented as mean ± SEM for n = 2, while in c, data are presented as mean ± SEM for n = 3