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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. 1. — T155 is essential for the apoptotic function of Par-4. (A) Schematic representation of NLS1, NLS2, S154, T155, and the SAC domain of full-length Par-4. PC-3 cells were transiently transfected with vector, Par-4, ΔNLS2, or the point mutant 154A or 155A; expression of Par-4 and mutants was examined by Western blot analysis with the Par-4 antibody or actin antibody (B). The ability of Par-4 or the mutants to induce apoptosis at 48 h posttransfection (C and D) was examined in PC-3 cells by either ICC with the Par-4 antibody and secondary antibody conjugated to Alexa Fluor 488 (green fluorescence) followed by nuclear staining with DAPI (D, left) or by staining with FITC-conjugated annexin V antibody (D, right). Cells expressing Par-4 or mutant proteins were scored for chromatin condensation or annexin V-positive cells indicative of apoptosis by confocal microscopy, as described in Materials and Methods. (C) Quantitative data. Nomarski images show total number of cells in each field (D, right). To determine inhibition of NF-κB transcriptional activity by Par-4 or its mutants (E), PC-3 cells were transfected for 48 h with NF-κB-luc reporter construct and β-galactosidase plasmid together with vector, Par-4, or mutant constructs. Whole-cell lysates were subjected to luciferase assays, and luciferase activity was normalized to the corresponding β-galactosidase activity. PC-3 cells were transiently transfected with expression constructs for GFP-SAC or GFP-SAC/155A mutant. Expression of the mutants was confirmed by Western blot analysis with the Par-4 or actin antibody (F), and the ability of the mutants to induce apoptosis was quantified by confocal microscopy (G and H). In the results shown in panel H, DAPI is pseudocolored red, and the yellow fluorescence resulting from the overlay with the GFP fusion proteins indicates nuclear localization. NIH 3T3/Ras cells were transiently transfected with the indicated expression constructs, and the ability of the constructs to induce apoptosis was quantified by confocal microscopy (I and J).

FIG. 1. — T155 is essential for the apoptotic function of Par-4. (A) Schematic representation of NLS1, NLS2, S154, T155, and the SAC domain of full-length Par-4. PC-3 cells were transiently transfected with vector, Par-4, ΔNLS2, or the point mutant 154A or 155A; expression of Par-4 and mutants was examined by Western blot analysis with the Par-4 antibody or actin antibody (B). The ability of Par-4 or the mutants to induce apoptosis at 48 h posttransfection (C and D) was examined in PC-3 cells by either ICC with the Par-4 antibody and secondary antibody conjugated to Alexa Fluor 488 (green fluorescence) followed by nuclear staining with DAPI (D, left) or by staining with FITC-conjugated annexin V antibody (D, right). Cells expressing Par-4 or mutant proteins were scored for chromatin condensation or annexin V-positive cells indicative of apoptosis by confocal microscopy, as described in Materials and Methods. (C) Quantitative data. Nomarski images show total number of cells in each field (D, right). To determine inhibition of NF-κB transcriptional activity by Par-4 or its mutants (E), PC-3 cells were transfected for 48 h with NF-κB-luc reporter construct and β-galactosidase plasmid together with vector, Par-4, or mutant constructs. Whole-cell lysates were subjected to luciferase assays, and luciferase activity was normalized to the corresponding β-galactosidase activity. PC-3 cells were transiently transfected with expression constructs for GFP-SAC or GFP-SAC/155A mutant. Expression of the mutants was confirmed by Western blot analysis with the Par-4 or actin antibody (F), and the ability of the mutants to induce apoptosis was quantified by confocal microscopy (G and H). In the results shown in panel H, DAPI is pseudocolored red, and the yellow fluorescence resulting from the overlay with the GFP fusion proteins indicates nuclear localization. NIH 3T3/Ras cells were transiently transfected with the indicated expression constructs, and the ability of the constructs to induce apoptosis was quantified by confocal microscopy (I and J).