Kinetics of Pol II phosphorylation during heat shock induction on Drosophila polytene chromosomes and localization of HSF, Ser2-P, and P-TEFb in the active hsp70 puff. Shown is the transgenic hsp70-lacZ gene, located at 9DE in Bg9 flies (23), precisely mapped by electron microscopy to the end of 9D (46). Chromosomes were prepared as previously described (21) and were stained with antibodies to HSF (red) and either Ser5-P (green) in panel A or Ser2-P (green) in panels B and C, as well as P-TEFb (blue) in panel C. Purple arrows indicate the direction of transcription. (A and B) Time course is as indicated. Images underwent three-dimensional restoration. (C) Chromosomes were heat shocked for 2.5 min. Merged images show the various combinations of HSF (H), Ser2-P (2), and P-TEFb (P) immunofluorescence, as indicated by the plus sign. The white arrow indicates where P-TEFb and Ser2-P expand into the puff, resolving from HSF. These images did not undergo three-dimensional restoration.