Journal: Cell reports

Article Title: Loss of Snf5 induces formation of an aberrant SWI/SNF complex

doi: 10.1016/j.celrep.2017.02.017

Figure Lengend Snippet: (A–B) Nucleosomes modified at residue 53 of histone H2B were used for monitoring movement of DNA on the octamer surface. DNA cleavage products were resolved on a denaturing 6% polyacrylamide gel and visualized by phosphorimaging. Numbers on the right side of the gel image refer to number of nucleotides (nt) moved from the starting cleavage position (0). Nucleosomes were remodeled with WT (A), and Δsnf5 SWI/SNF (B) for 0, 5, 10, 20, 30, 40, 50, 60, 70, 80 and 160 s using 4.4 μM ATP. (C) The amount of DNA cleaved at starting position (0) was plotted versus time for WT SWI/SNF and Δsnf5 SWI/SNF. Rate constants (k) obtained by fitting data to single exponential function were 0.044± 0.004 s−1 (WT) and 0.016 ± 0.005 s−1 (Δsnf5). Bars and reported values are the mean ± SE from two replicates. (D) ATPase assays were performed in the same conditions as (A and B) and the released radioactive phosphate (Pi) was separated from non-hydrolyzed ATP by thin layer chromatography (TLC). The percentage Pi released is plotted as a function of time. ATP hydrolysis rates obtained were 0.9 μM s−1 (WT) and 0.7 μM s−1 (Δsnf5).

Article Snippet: Normalized band intensities were plotted as a function of time and fitted into single phase exponential equation using GraphPad (PRISM Version 6.0b).

Techniques: Modification, Residue, Thin Layer Chromatography

Journal: PLoS ONE

Article Title: Recombinant hTRBP and hPACT Modulate hAgo2-Catalyzed siRNA-Mediated Target RNA Cleavage In Vitro

doi: 10.1371/journal.pone.0146814

Figure Lengend Snippet: Representative stopped-flow graphs are shown. The inserts show the reaction on a shorter time scale. 50 nM fluorescently labeled double-stranded siRNA (aslam-FAM/slam) were rapidly mixed with 500 nM hTRBP (A) or hPACT (B). Data were fitted to a double exponential equation yielding the following rates: k 1 : 11.1 (± 2.3) s -1 and k 2 : 0.02 (± 0.001) s -1 for hTRBP and k 1 : 37.8 (± 2.5) s -1 and k 2 : 0.04 (± 0.005) s -1 for hPACT.

Article Snippet: Data were fitted using a single exponential equation (Grafit 5, Erithacus Software).

Techniques: Labeling

Journal: PLoS ONE

Article Title: Recombinant hTRBP and hPACT Modulate hAgo2-Catalyzed siRNA-Mediated Target RNA Cleavage In Vitro

doi: 10.1371/journal.pone.0146814

Figure Lengend Snippet: The different substrate combinations (ss-siRNA & long target, ds-siRNA & long target, ds-siRNA or ss-siRNA & short target) are depicted as little cartoons on top of the corresponding graph. For target RNA cleavage either 2.5 nM radioactively labeled ICAM-1-IVT (A, B) or s2b (D) were mixed with binary complexes consisting of either 100 nM as2b (A, D) or as2b/s2b (B) and 3 μM hAgo2 in the absence (open circles) or presence of 3 μM hTRBP (closed circles), hTRBP-D12 (open squares) and hPACT (closed squares), respectively. For ds-siRNA cleavage, i.e. passenger cleavage (C), 30 nM 5’-32P-passenger labeled siRNA (as2b/s2b) was mixed with 3 μM hAgo2 in the presence or absence of dsRBPs as described above. Samples were taken at different time points, analyzed by denaturing PAGE (8% for ICAM-1-IVT and 20% for s2b) and detected by autoradiography (Figure H in ). Data shown are averaged from at least three independent measurements. Error bars represent standard deviation. Experimental data were fitted to an exponential equation. Rate constants and corresponding amplitudes are listed in .

Article Snippet: Data were fitted using a single exponential equation (Grafit 5, Erithacus Software).

Techniques: Labeling, Autoradiography, Standard Deviation

Journal: PLoS ONE

Article Title: Recombinant hTRBP and hPACT Modulate hAgo2-Catalyzed siRNA-Mediated Target RNA Cleavage In Vitro

doi: 10.1371/journal.pone.0146814

Figure Lengend Snippet: Representative stopped-flow graphs are shown. Ternary complexes composed of 500 nM hAgo2, 20 nM guide RNA (as2b-FAM) and 40 nM target RNA (s2b) were preassembled and subsequently rapidly mixed with 2 μM unlabeled guide RNA and 500 nM hTRBP-D12 (A) or hTRBP (B). In both cases data could be best fitted using a triple exponential equation, yielding the following rate constants: (A) k -1 : 18.4 (± 1.2) s -1 , k -2 : 0.1 (± 0.007) s -1 , k -3 : 0.002 (± 0.0005) s -1 and (B) k -1 : 15.9 (± 4.6) s -1 , k -2 : 0.05 (± 0.001) s -1 , k -3 : 0.0005 (± 0.0002) s -1 . Rate constants are summarized in .

Article Snippet: Data were fitted using a single exponential equation (Grafit 5, Erithacus Software).

Techniques:

Journal: PLoS ONE

Article Title: Recombinant hTRBP and hPACT Modulate hAgo2-Catalyzed siRNA-Mediated Target RNA Cleavage In Vitro

doi: 10.1371/journal.pone.0146814

Figure Lengend Snippet: Representative stopped-flow graphs are shown. The inserts show the reaction on a shorter time scale. 20 nM of fluorescently labeled double-stranded siRNA (as2b-FAM/s2B) were pre-incubated with 600 nM hTRBP (A, C) or hPACT (B, D). The binary complexes were subsequently rapidly mixed with either hAgo2 (400 nM in case of A and 600 nM in case of B) or 600 nM hAgo2-PAZ9 (C, D). Data were fitted to an exponential equation. For hAgo2 & hTRBP: k 1 : 28.8 (± 10.8) s -1 , k 2 : 0.33 (±0.03) s -1 and k 3 : 0.002 (± 0.0001) s -1 and for hAgo2 & hPACT: k 1 : 18.6 (± 3.5) s -1 , k 2 : 0.13 (± 0.02) s -1 and k 3 : 0.01 (± 0.001) s -1 were determined. With the mutant hAgo2-PAZ9 the following rate constant were calculated: k 1 : 14.9 (± 2.7) s -1 , k 2 : 0.07 (±0.01) s -1 and k 3 : 0.01 (± 0.0009) s -1 in the presence of hTRBP and k 1 : 16.2 (± 0.6) s -1 , k 2 : 0.38 (±0.01) s -1 and k 3 : 0.007 (± 0.002) s -1 in the presence of hPACT.

Article Snippet: Data were fitted using a single exponential equation (Grafit 5, Erithacus Software).

Techniques: Labeling, Incubation, Mutagenesis