Journal: Molecular and Cellular Biology

Article Title: Protein Phosphatase 2A Activity Affects Histone H3 Phosphorylation and Transcription in Drosophila melanogaster

doi: 10.1128/mcb.23.17.6129-6138.2003

Figure Lengend Snippet: FIG. 2. Protein phosphatase type 2A dephosphorylates Ser10- phosphorylated histone H3 both in vivo and in vitro. (A) Purified core histones containing Ser10-phosphorylated histone H3 were incubated with purified PP2A in the presence or absence of 50 nM okadaic acid as indicated. The PP2A enzyme strongly dephosphorylates Ser10- phosphorylated histone H3. Catalytic activity of PP2A is strongly in- hibited by incubation with 50 nM okadaic acid. (B) Whole-larval ex- tracts were prepared from wild-type and twsP mutant larvae that either were not heat shocked (0) or were heat shocked for 20 min (20), resolved on an SDS–15% polyacrylamide gel, and immunostained for the presence of the Ser10-phosphorylated isoform of histone H3. The twsP mutant extracts contained a greater concentration of Ser10-phos- phorylated histone H3 than wild-type controls both before and after heat shock.

Article Snippet: Briefly, 5 g of core histones prepared from Colcemid-treated HeLa cells (Upstate Biotechnology) was used as a substrate in assay buffer (10 mM HEPES [pH 7.0], 1 mM dithiothreitol, 1 mM MnCl2, 10 g of BSA/ml, 50 M leupeptin) containing 0.1 U of purified PP2A AC dimer (Upstate Biotechnology).

Techniques: In Vivo, In Vitro, Incubation, Activity Assay, Mutagenesis, Concentration Assay

Journal: Molecular and Cellular Biology

Article Title: Protein Phosphatase 2A Activity Affects Histone H3 Phosphorylation and Transcription in Drosophila melanogaster

doi: 10.1128/mcb.23.17.6129-6138.2003

Figure Lengend Snippet: FIG. 6. The SET oncoprotein inhibits PP2A-mediated dephosphorylation of phosphohistone H3 and is present at transcriptionally active loci. (A) Purified human core histones containing Ser10-phosphorylated histone H3 (lane 1) were incubated with purified human PP2A (lane 2). The presence of 1 l of in vitro reticulocyte lysate-translated SET is capable of inhibiting PP2A-mediated dephosphorylation of histone H3 (lane 4). Translation reactions that did not contain SET protein showed minimal inhibition of PP2A activity (lane 3). (B) Antibodies to the SET gene product were generated and used to detect SET in whole Drosophila pupa extracts (lane 1) and the in vitro-translated SET protein expressed via a reticulocyte lysate system and utilized in a PP2A activity assay (lanes 3 and 4). Lane 3 contains 5 l of reticulocyte lysate that does not contain the expressed SET protein. Lane 4 contains 5 l of reticulocyte lysate containing expressed SET. Lane 2 shows the Coomassie staining of lane 1. Lanes 5 and 6 show Coomassie staining of lanes 3 and 4, respectively. Molecular mass size standards (in kilodaltons) are indicated at right. (C,D, and E) Genomic distribution of SET protein in polytene chromosomes prepared from wild-type larvae before (C) and after (E) heat shock and immunostained for the presence of SET (SET, red) and the Ser10-phosphorylated histone H3 isoform (phospho-H3, green). Chromosome spreads were also counterstained with DAPI (white) to view the DNA. DAPI counterstain information is represented as blue in merged images. Before heat shock, the SET protein is detected at puffed and H3-phosphorylated (transcriptionally active) loci, including the ecdysone-regulated genes, which are labeled (C). In situ hybridization with a fragment of the hsp70 transcription unit (hsp70, green) reveals that prior to heat shock, the SET protein (SET, red) does not colocalize with the hsp70 genes (D). During heat shock, strong SET staining is also detected at the actively transcribing heat shock puffs (E).

Article Snippet: Briefly, 5 g of core histones prepared from Colcemid-treated HeLa cells (Upstate Biotechnology) was used as a substrate in assay buffer (10 mM HEPES [pH 7.0], 1 mM dithiothreitol, 1 mM MnCl2, 10 g of BSA/ml, 50 M leupeptin) containing 0.1 U of purified PP2A AC dimer (Upstate Biotechnology).

Techniques: De-Phosphorylation Assay, Incubation, In Vitro, Inhibition, Activity Assay, Generated, Staining, Labeling, In Situ Hybridization