Fig 2: Pin1 mutant is defective in the oxidative stress induced transcription response at the initiation to elongation transition. (A) Whole-cell protein extracts of the strains indicated were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS-PAGE analyzed phosphorylation by western immunoblotting with CTD phospho-specific antibodies. Antibody against a-tubulin was used as the control. The relative level of phosphorylated protein was shown in (B). Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance (P<0.05, t-test unpaired) verse wild type. (C) ChIP assays showing the recruitment of Rpb1 to the promoter and the gene coding regions (ORF) of ctt1, gpd1 and hsp9 genes upon oxidative stress in wild type cells and pin1? mutant. Samples were prepared from the indicated strains treated with 2 mM H2O2 for the time points indicated. The DNA recovered from the IP was amplified using quantitative PCR and SYBR green with primers specific to the promoter of the indicated genes normalized to that of the non-stress responsive cdc2 gene. Data were expressed as means ± SD in triplicate. Asterisks indicated statistical significance (P< 0.05, t-test unpaired) verse wild type. (D) ChIP assays showing the recruitment of pSer5-Rpb1 to the promoters of ctt1, gpd1 and hsp9 genes upon oxdative stress in wild type cells and pin1? mutant as in (C). Data were expressed as means ± SD in triplicate. Asterisks indicated statistical significance (P< 0.05, t-test unpaired) verse wild type.

Fig 3: A mutual exclusive binding mode of Rpb1 to Pin1 over Sty1. (A) Recombinant proteins of Sty1 and CTD-GFP from yeast cells were subjected to GFP pulldown assay in the presence or absence of Pin1 followed by Western immunoblotting analysis with antibodies indicated. *Degradation product of Pin1-GST. The relative level of Sty1 pull down by CTD-GFP was shown. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance (P<0.05, t-test unpaired) verse control without Pin1. (B) Whole-cell protein extracts of the strains indicated were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS-PAGE and subjected to immunoblotting using anti-His antibodies to reveal Pin1 proteins. Antibody against a-tubulin was used as the control. (C) Tenfold serial dilutions of strains indicated were spotted onto EMM+thiamine agar plates without (control) or with 2 mM H2O2 and photographed after 3 days incubation at 30°C. (D) Coimmunoprecipitation was performed with extracts prepared from Sty1-GFP tagged strains treated with 0.5 M NaCl for the time point indicated. GFP-Trap affinity resin was used to pull down GFP proteins. Immunoprecipitates were analyzed by Western immunoblotting using antibodies against GFP and Rpb1-CTD. The relative level of Rpb1 pull down by Sty1 was shown. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance (P< 0.05, t-test unpaired) verse control of no treatment. (E) Tenfold serial dilutions of strains indicated were spotted onto YES-agar plates without (control) or with 200 mM NaCl and photographed after 3 days incubation at 30°C.

Fig 4: Sty1 interacted and phosphorylated Rpb1-CTD at Ser5. (A) Coimmunoprecipitation was performed with extracts prepared from indicated strains treated with 2 mM H2O2 for 30 min. GFP-Trap affinity resin was used to pull down GFP proteins. Immunoprecipitates were analyzed by Western immunoblotting using antibodies against GFP and Rpb1-CTD. The relative level of Rpb1 pull down by Sty1 and its phosphorylation status was shown in (B). Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance (P< 0.05, t-test unpaired) verse wild type. (C) Coimmunoprecipitation was performed with extracts prepared from Pin1-GFP tagged strains treated with 2 mM H2O2 for 30 min. GFP-Trap affinity resin was used to pull down GFP proteins. Immunoprecipitates were treated with/without CIP and analyzed by Western immunoblotting using antibodies against GFP and Rpb1-CTD. *Degradation product of Pin1-GFP. The relative level of Rpb1 pull down by Pin1 was shown. Data were expressed as means±SD in triplicate. Asterisks indicated statistical significance (P<0.05, t-test unpaired) verse control of no treatments. (D, E) Purified GST-CTD from Escherichia coli and recombinant protein of Sty1 from yeast cells were subjected to GST pulldown assay followed by Western immunoblotting analysis with antibodies against GST and His. (F) In vitro kinase assay using Sty1-His extracted and immunoprecipitated from activated cells. Following incubation with GST-CTD, proteins were resolved by SDS-PAGE and probed with antibodies indicated.

Fig 5: Pin1 mutant is not defective in MAPK-mediated signal transduction pathway. (A) Whole-cell protein extracts of the strains indicated were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS-PAGE and subjected to Western immunoblotting using anti-HA antibodies to reveal proteins of interest. Anti p-p38 antibody cross-reacted with phosphorylated Sty1. Antibody against a-tubulin was used as the loading control. (B) ChIP assays showing recruitment of Atf1-myc to the promoter of ctt1, gpd1, hsp9 and cdc2 genes upon oxdative stress. The latter was not induced upon stress. Samples were prepared from atf1-myc and atf1-myc pin1? cells treated with 2 mM H2O2 for the time points indicated. The DNA recovered from the IP was assayed by PCR using primers specific to the promoter of genes indicated. The control lanes show DNA amplified from whole cell extracts (WCE) prior to performing the IP. (C) ChIP assays showing recruitment of Atf1-myc and Sty1-myc to the promoter of ctt1, gpd1 and hsp9 genes upon oxdative stress in pin1? mutant and wild type cells. Samples were prepared from strains indicated treated with 2 mM H2O2 for the time points indicated. The DNA recovered from the IP was amplified using quantitative PCR and SYBR green with primers specific to the promoter of genes indicated normalized to that of the non-stress responsive cdc2 gene. Data were expressed as means ± SD in triplicate. Asterisks indicated statistical significance (P< 0.05, t-test unpaired) verse wild type.