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Fig. 5 | Epigenetics & Chromatin

Fig. 5

From: Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus

Fig. 5

HSF recruitment to chromatin is necessary and sufficient for the dynamic protein–protein interaction between Pho and Spt5. a ChIP-qPCR measurements for the occupancy levels of HSF at the hsp70 locus in S2 DRSC cells upon HS (heat shock), radicicol, FP + HS (flavopiridol + heat shock) and Na Sal (Sodium Salicylate) treatment conditions, respectively. In case of radicicol treatment, the control condition was DMSO treatment. For FP + HS, the control condition was FP control and for Na Sal, the control condition was nuclease-free water. Distance of the location of the primers used from the hsp70 TSS is − 85 bp and has been depicted in the form of a cartoon below the data figure. b qRT-PCR measurements of hsp70 transcript levels in S2 DRSC cells under HS, radicicol, FP + HS and Na Sal treatment conditions, respectively. Distance of the location of the primers used from the hsp70 TSS is + 1492 bp and has been depicted in the form of a cartoon below the data figure. c co-IP assays of S2 DRSC cells transiently transfected with plasmids expressing FLAG-tagged Spt5 (FLAG-Spt5) and HA-tagged Pho (HA-Pho). The S2 DRSC cells were subjected to HS, radicicol, FP + HS and Na Sal, respectively; cell lysates were used for pull-downs with an anti-FLAG antibody and later probed by Western blot with an anti-HA antibody. d Quantification of the co-IP assays in c. Details of the procedure used for quantification can be found in Materials and Methods. e co-IP assays of S2 DRSC cells transiently transfected with plasmids expressing FLAG-tagged Spt5 (FLAG-Spt5) and HA-tagged Pho (HA-Pho). Cells were either treated with HSF or LacZ RNAi for 4 days, then either maintained at control conditions or heat shocked, cell lysates were prepared and used for pull-downs using an anti-FLAG antibody and later probed by Western blot using an anti-HA antibody. f qRT-PCR measurements of hsp70 transcript levels in S2 DRSC cells under HSF and LacZ RNAi conditions. Distance of the location of the primers used from the hsp70 TSS is + 1492 bp and has been depicted in the form of a cartoon below the data figure. Data information: In a and b, data are presented as mean ± SEM for n = 2, while in e, data are presented as mean ± SEM for n = 3

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