Journal: Journal of Biological Chemistry

Article Title: Dynamics of the Ternary Complex Formed by c-Myc Interactor JPO2, Transcriptional Co-activator LEDGF/p75, and Chromatin

doi: 10.1074/jbc.m113.525964

Figure Lengend Snippet: FIGURE 1. JPO2 interacts dynamically with chromatin. A, schematic representation of JPO2 with indication of the known/predicted chromatin and protein- protein interaction domains. B, Western blot of HeLa cells expressing eGFP-JPO2, 24 h post-transfection. The eGFP-JPO2 protein was detected using an eGFP antibody. Non-transfected cells were used as a negative control. C–E, confocal imaging, CP, and FCS analysis of HeLap75KD cells expressing eGFP-JPO2. C, both interphase(top)andmitotic(bottom)cellswereimagedafterstainingforDNAwithDAPI.D,CPcurveswerefittedwithEquation1(solidline).Errorbars,S.D.after normalization. E, average FCS autocorrelation functions were fitted with Equation 2 (solid line). Error bars, S.D. on the normalized data after fitting. D and E, CP and FCS data in HeLap75KD cells expressing eGFP or eGFP-LEDGF/p75 are shown for comparison (21). Parameters after fitting are given in Table 1. LZ, leucine zipper; NLS, nuclear localization sequence; au, arbitrary units.

Article Snippet: Transfections—Transient expression was achieved via cationic lipid-mediated plasmid DNA transfection (Lipofectamine 2000, Invitrogen).

Techniques: Western Blot, Expressing, Transfection, Negative Control, Imaging, Comparison, Sequencing

Journal: Journal of Biological Chemistry

Article Title: Dynamics of the Ternary Complex Formed by c-Myc Interactor JPO2, Transcriptional Co-activator LEDGF/p75, and Chromatin

doi: 10.1074/jbc.m113.525964

Figure Lengend Snippet: FIGURE 2. Dynamic chromatin interaction of JPO2 is enhanced by LEDGF/p75. A, schematic representation of LEDGF/p75 with indication of the known chromatin and protein-protein interaction domains. B, Western blot of HeLa cells expressing mRFP-LEDGF/p75, 24 h post-transfection. Endogenous LEDGF/ p75 (L) and mRFP-LEDGF/p75 (mL) were detected using a LEDGF/p75-specific antibody. The minor band below the mRFP-LEDGF/p75 band is due to partial intrachain cleavage of the mRFP-moiety (64). Non-transfected cells were used as a negative control. C and D, confocal imaging and FCS analysis of HeLap75KD cells expressing mRFP-LEDGF/p75. C, both interphase (top) and mitotic (bottom) cells were imaged after staining for DNA with DAPI. D, FCS analysis of eGFP- and mRFP-tagged LEDGF/p75. Average autocorrelation functions and fit with Equation 2 (solid line). Error bars, S.D. on the normalized data after fitting. Parameters after fitting are depicted in Table 1. The differences between the eGFP- and mRFP-LEDGF/p75 autocorrelation functions in the 10–1000-s time scale are due to the strong blinking contribution in the mRFP1 autocorrelation function (52). E–H, confocal imaging, CP, FCS, and FCCS analysis of HeLap75KD cells co-expressing eGFP-JPO2 and mRFP-LEDGF/p75. E, both interphase (top) and mitotic (bottom) cells were imaged after staining for DNA with DAPI. F, normalizedaverageFCCScross-correlationfunctionfromcellswithequimolarexpressionofeGFP-andmRFP-labeledproteins.Errorbars,S.E.G,CPcurveswere fitted with Equation 1 (solid line). Error bars, S.D. after normalization. H, average FCS autocorrelation functions were fitted with Equation 2 (solid line). Error bars, S.D. on the normalized data after fitting. G and H, CP and FCS data in HeLap75KD cells expressing eGFP-LEDGF/p75 (21) and/or eGFP-JPO2 (Fig. 1, D and E) are shown for comparison. Parameters after fitting are depicted in Table 1. au, arbitrary units.

Article Snippet: Transfections—Transient expression was achieved via cationic lipid-mediated plasmid DNA transfection (Lipofectamine 2000, Invitrogen).

Techniques: Western Blot, Expressing, Transfection, Negative Control, Imaging, Staining, Comparison

Journal: Journal of Biological Chemistry

Article Title: Dynamics of the Ternary Complex Formed by c-Myc Interactor JPO2, Transcriptional Co-activator LEDGF/p75, and Chromatin

doi: 10.1074/jbc.m113.525964

Figure Lengend Snippet: FIGURE 4. LEDGF/p75 trails JPO2 along chromatin via its PWWP domain. A, Western blot of HeLa cells expressing mRFP-LEDGF/p75 (WT) or chromatin binding-deficient mRFP-LEDGF/p75 mutant (K56D/R74D), 24 h post-transfection. Endogenous LEDGF/p75 (L) and mRFP-LEDGF/p75 (mL) were detected using a LEDGF/p75 specific antibody. B–F, confocal imaging, CP, FCS, and FCCS analysis of HeLap75KD cells expressing mRFP-LEDGF/p75K56D/R74D or eGFP-JPO2 mRFP-LEDGF/p75K56D/R74D.BandC,bothinterphase(top)andmitotic(bottom)cellswereimagedafterstainingforDNAwithDAPI.D,CPcurveswerefittedwith Equation 1 (solid line). Error bars, S.D. after normalization. E, average FCS autocorrelation functions were fitted with Equation 2 (solid line). Error bars, S.D. on the normalized data after fitting. D and E, CP and FCS data in HeLap75KD cells expressing eGFP-JPO2 (Fig. 1D) or eGFP-JPO2 mRFP-LEDGF/p75 (Fig. 2, G and H) are shown for comparison. Parameters after fitting are given in Table 1. F, box plot of the CCrel values from different cells (number of cells 6) with equimolar expression of eGFP- and mRFP-labeled proteins. Whiskers represent the 5th and 95th percentile. *, statistically significant difference (p 0.01 from an unpaired Student’s t test). FCCS data from Fig. 2F are shown for comparison. au, arbitrary units.

Article Snippet: Transfections—Transient expression was achieved via cationic lipid-mediated plasmid DNA transfection (Lipofectamine 2000, Invitrogen).

Techniques: Western Blot, Expressing, Binding Assay, Mutagenesis, Transfection, Imaging, Comparison, Labeling