Journal: Epigenetics & Chromatin

Article Title: Heterogeneity in the kinetics of nuclear proteins and trajectories of substructures associated with heterochromatin

doi: 10.1186/1756-8935-4-5

Figure Lengend Snippet: Cellular patterns of selected proteins . The dynamics of selected proteins were studied using GFP technology combined with fluorescence recovery after photobleaching (FRAP). The following proteins were analysed: heterochromatin protein 1 (HP1α) (a1), (HP1β) (a2), B lymphoma Mo-MLV insertion region 1 (BMI1) (b), telomeric-repeat binding factor 1 (TRF1) (c), RNA polymerase I large subunit (RPA194) (d), upstream binding factor (UBF) (e), histones H2B (f1), H4 (f2), ubiquitin (Ub) (g), A-type lamins (h), histone demethylase JMJD2b (i), tumour suppressor p53 (j), oncoprotein c-MYC (k), β-catenin (l), STAT1 (m), α-tubulin (n), PML protein (o), and Oct3/4 (p).

Article Snippet: The following plasmids were used in this study: JMJD2b-GFP (obtained from Professor Thomas Jenuwein and Dr Nicholas Shukeir, Max-Planck Institute of Immunobiology, Freiburg, Germany); HP1α-GFP and HP1β-GFP (from the laboratory of Dr Tom Misteli, National Institutes of Health, Bethesda, MD, USA); RPA194-GFP (Addgene Inc., Cambridge, MA, USA; #17660); UBF-GFP (Addgene; #17656); Ubiquitin-GFP (Addgene; #11928); p53-GFP (Addgene; #12091); pmCherry-alpha-tubulin-IRES-puro2 (Addgene; #1360); lamin A-GFP (Addgene; #17662); c-myc-GFP (a gift from Dr Hiroyoshi Ariga, Hokkaido University, Graduate School of Pharmaceutical Sciences, Kita-Ku, Sapporo, Japan), β-catenin-enhanced GFP and STAT1-GFP (gifts from Dr Vítězslav Bryja and Dr Jiří Pacherník, Faculty of Sciences, Masaryk University, Brno, Czech Republic).

Techniques: Fluorescence, Binding Assay, Ubiquitin Proteomics

Journal: Epigenetics & Chromatin

Article Title: Heterogeneity in the kinetics of nuclear proteins and trajectories of substructures associated with heterochromatin

doi: 10.1186/1756-8935-4-5

Figure Lengend Snippet: Test of correlations between molecular weight and recovery time after photobleaching . (a) The kinetic properties after photobleaching were studied for the following proteins: heterochromatin protein 1α (HP1α), HP1β, B lymphoma Mo-MLV insertion region 1 (BMI1), telomeric-repeat binding factor 1 (TRF1), RNA polymerase I large subunit (RPA194), upstream binding factor (UBF), H2B, H4, ubiquitin (Ub), lamin A (central), JMJD2b, p53, c-MYC, β-catenin, STAT1, α-tubulin, PML, and Oct3/4. α-Tubulin was tagged with mCherry (35 kDa) and other proteins were tagged with GFP (28 kDa). Molecular weights of individual proteins were compared by correlation analysis with the level of relative fluorescence 6 s after photobleaching [ R 6 ]. (b) A correlation between molecular weight and fluorescence recovery of proteins was found for proteins that were evenly dispersed throughout the nucleoplasm: HP1α, HP1β, BMI1, TRF1, H2B, H4, Ub, lamin A (central), JMJD2B, p53, c-MYC, β-catenin, STAT1, α-tubulin, Oct3/4. (c) No correlation between molecular weight and fluorescence recovery for proteins that were accumulated into foci: HP1α, HP1β, BMI1, TRF1, and PML. (d) No correlation was detected between molecular weight and fluorescence recovery for proteins accumulated into nucleoli: HP1β, RPA194, UBF. Pearson's correlation coefficient for (n-2) = 17 is 0.456; for (n-2) = 13 is 0.514; for (n-2) = 3 is 0.878 and for (n-2) = 1 is 0.997. These values are for α = 0.05. Regression lines surrounded by 95% confidence intervals (dashed curves) are shown in all panels except panel (d); to unify the axis scale, confidence intervals are not shown.

Article Snippet: The following plasmids were used in this study: JMJD2b-GFP (obtained from Professor Thomas Jenuwein and Dr Nicholas Shukeir, Max-Planck Institute of Immunobiology, Freiburg, Germany); HP1α-GFP and HP1β-GFP (from the laboratory of Dr Tom Misteli, National Institutes of Health, Bethesda, MD, USA); RPA194-GFP (Addgene Inc., Cambridge, MA, USA; #17660); UBF-GFP (Addgene; #17656); Ubiquitin-GFP (Addgene; #11928); p53-GFP (Addgene; #12091); pmCherry-alpha-tubulin-IRES-puro2 (Addgene; #1360); lamin A-GFP (Addgene; #17662); c-myc-GFP (a gift from Dr Hiroyoshi Ariga, Hokkaido University, Graduate School of Pharmaceutical Sciences, Kita-Ku, Sapporo, Japan), β-catenin-enhanced GFP and STAT1-GFP (gifts from Dr Vítězslav Bryja and Dr Jiří Pacherník, Faculty of Sciences, Masaryk University, Brno, Czech Republic).

Techniques: Molecular Weight, Binding Assay, Ubiquitin Proteomics, Fluorescence