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. 2005 Feb;25(3):1146–1161. doi: 10.1128/MCB.25.3.1146-1161.2005

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Copyright © 2005, American Society for Microbiology

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FIG. 5. — Endogenous Par-4 induces T155- and PKA-dependent apoptosis. To study localization of Par-4 and apoptosis in response to cAMP and doxorubicin treatment (A and C), MEFs were treated with vehicle, 10 μM 8-Cl-cAMP, 100 nM doxorubicin, 8-Cl-cAMP plus doxorubicin, or 8-Cl-cAMP plus 20 μM PKI and subjected to ICC analysis with Par-4 antibody followed by FITC-conjugated secondary antibody (green). The nuclei were stained with DAPI (pseudocolored red) and visualized under a confocal microscope (A) and scored for apoptosis (A and C). To study phosphorylation of T155 by cAMP (B), MEFs were treated with 10 μM cAMP, cAMP plus PKI 20 μM, or cAMP plus 40 μM PKI; whole-cell lysates were subjected to Western blot analysis with pT155, Par-4, pCREB, total CREB, or actin antibodies (B, top). PKA activity was determined in the lysates with the cAMP-dependent PKA Signatect assay kit (B, bottom). To determine whether vincristine induces T155 phosphorylation of Par-4 (D), HEL cells were treated with vehicle or 100 nM vincristine for 24 and 48 h, and Western blot analysis was performed on the whole-cell lysates with pT155, Par-4, or actin antibodies. To study vincristine-inducible apoptosis (E), HEL cells were treated with vehicle or vincristine in the presence or absence of 20 or 40 μM PKI peptide or transiently transfected with vector or GFP-Par-4 155A plasmid and assayed for apoptosis as described in the legend to Fig. 1. To determine whether inhibition of Par-4 or PKA expression inhibits apoptosis by vincristine (F), HEL cells were transiently transfected with 10 μM nonspecific control siRNA, siRNA for Par-4, or siRNA for PKA α plus β duplexes, treated with either vehicle or 100 nM vincristine, and assayed for apoptosis as described in the legend to Fig. 1. Whole-cell lysates from the siRNA-transfected cells were subjected to Western blot analysis with antibodies against PKAc α and β, Par-4, and actin (G). To determine the effect of the various treatments on PKA activity, whole-cell lysates prepared from the cells after various treatments, as described above (D and E), were tested for PKA enzymatic assay with the cAMP-dependent PKA Signatect assay kit (H).

FIG. 5. — Endogenous Par-4 induces T155- and PKA-dependent apoptosis. To study localization of Par-4 and apoptosis in response to cAMP and doxorubicin treatment (A and C), MEFs were treated with vehicle, 10 μM 8-Cl-cAMP, 100 nM doxorubicin, 8-Cl-cAMP plus doxorubicin, or 8-Cl-cAMP plus 20 μM PKI and subjected to ICC analysis with Par-4 antibody followed by FITC-conjugated secondary antibody (green). The nuclei were stained with DAPI (pseudocolored red) and visualized under a confocal microscope (A) and scored for apoptosis (A and C). To study phosphorylation of T155 by cAMP (B), MEFs were treated with 10 μM cAMP, cAMP plus PKI 20 μM, or cAMP plus 40 μM PKI; whole-cell lysates were subjected to Western blot analysis with pT155, Par-4, pCREB, total CREB, or actin antibodies (B, top). PKA activity was determined in the lysates with the cAMP-dependent PKA Signatect assay kit (B, bottom). To determine whether vincristine induces T155 phosphorylation of Par-4 (D), HEL cells were treated with vehicle or 100 nM vincristine for 24 and 48 h, and Western blot analysis was performed on the whole-cell lysates with pT155, Par-4, or actin antibodies. To study vincristine-inducible apoptosis (E), HEL cells were treated with vehicle or vincristine in the presence or absence of 20 or 40 μM PKI peptide or transiently transfected with vector or GFP-Par-4 155A plasmid and assayed for apoptosis as described in the legend to Fig. 1. To determine whether inhibition of Par-4 or PKA expression inhibits apoptosis by vincristine (F), HEL cells were transiently transfected with 10 μM nonspecific control siRNA, siRNA for Par-4, or siRNA for PKA α plus β duplexes, treated with either vehicle or 100 nM vincristine, and assayed for apoptosis as described in the legend to Fig. 1. Whole-cell lysates from the siRNA-transfected cells were subjected to Western blot analysis with antibodies against PKAc α and β, Par-4, and actin (G). To determine the effect of the various treatments on PKA activity, whole-cell lysates prepared from the cells after various treatments, as described above (D and E), were tested for PKA enzymatic assay with the cAMP-dependent PKA Signatect assay kit (H).