American Society of Hematology

Figure 3

$Figure 3. PDLIM2 targets Tax into the nuclear matrix for proteasomal degradation. (A) 293 cells transfected with Tax in the presence of increasing amounts of PDLIM2 were left untreated or treated with MG132 for 3 hours, followed by cell fractions and IB to detect expression levels of the indicated proteins. The ratio of cytoplasm–soluble nuclear fraction–insoluble nuclear fraction used for IB assay was 1:2:2. The Tax levels were also quantitated by densitometry. (B) Hela cells transfected with Tax alone or together with PDLIM2 were analyzed by indirect immunofluorescence to visualize the indicated proteins. Tax was shown in green. SC-35, PML, and the proteasome were shown in red. Original magnification, ×1000. (C) C8166 or MT-4 cells stably expressing an empty vector (top panel) or PDLIM2 (middle and bottom panels) were analyzed by confocal microscopy to visualize the proteasome (red) and Tax (green). The cells in the bottom panel were treated with MG132 for 3 hours prior to the immunofluorescence assays. Bar represents 10 μM. (D) 293 cells were transfected with Tax alone or together with increasing amounts of PDLIM2, followed by nuclear matrix extraction and IB. The percentiles of Tax in the nuclear matrix (NM) among total Tax proteins in the cells were presented. Error bars indicate standard deviations (n = 3). (E) 293 cells transfected with Tax alone or together with PDLIM2 were treated with MG132 or DMSO for 3 hours, followed by cell fractions as described in “Subcellular fractionation, immunoblotting, and immunoprecipitation assays.” The data presented the percentiles of MG132-recovered Tax in the cytoplasm (Cy), nucleoplasm (Nu), chromatin (Ch), and nuclear matrix (NM) based on the Tax amounts in the respective fractions from the DMSO-treated cells. Error bars indicate standard deviations (n = 3).$

PDLIM2 targets Tax into the nuclear matrix for proteasomal degradation. (A) 293 cells transfected with Tax in the presence of increasing amounts of PDLIM2 were left untreated or treated with MG132 for 3 hours, followed by cell fractions and IB to detect expression levels of the indicated proteins. The ratio of cytoplasm–soluble nuclear fraction–insoluble nuclear fraction used for IB assay was 1:2:2. The Tax levels were also quantitated by densitometry. (B) Hela cells transfected with Tax alone or together with PDLIM2 were analyzed by indirect immunofluorescence to visualize the indicated proteins. Tax was shown in green. SC-35, PML, and the proteasome were shown in red. Original magnification, ×1000. (C) C8166 or MT-4 cells stably expressing an empty vector (top panel) or PDLIM2 (middle and bottom panels) were analyzed by confocal microscopy to visualize the proteasome (red) and Tax (green). The cells in the bottom panel were treated with MG132 for 3 hours prior to the immunofluorescence assays. Bar represents 10 μM. (D) 293 cells were transfected with Tax alone or together with increasing amounts of PDLIM2, followed by nuclear matrix extraction and IB. The percentiles of Tax in the nuclear matrix (NM) among total Tax proteins in the cells were presented. Error bars indicate standard deviations (n = 3). (E) 293 cells transfected with Tax alone or together with PDLIM2 were treated with MG132 or DMSO for 3 hours, followed by cell fractions as described in “Subcellular fractionation, immunoblotting, and immunoprecipitation assays.” The data presented the percentiles of MG132-recovered Tax in the cytoplasm (Cy), nucleoplasm (Nu), chromatin (Ch), and nuclear matrix (NM) based on the Tax amounts in the respective fractions from the DMSO-treated cells. Error bars indicate standard deviations (n = 3).

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