storm phosphorimager analysis  (GraphPad Software Inc)

Journal:

Article Title: DNA Recognition by the Aberrant Retinoic Acid Receptors Implicated in Human Acute Promyelocytic Leukemia 1

doi:

Figure Lengend Snippet: DNA binding by RARα, PML-RARα, PLZF-RARα, or RXRα as characterized by electrophoretic mobility shift assay. RARα, PML-RARα, PLZF-RARα, and/or RXRα, isolated from baculovirus-infected Sf9 cells, were incubated with the radiolabeled consensus oligonucleotide presented in Fig. 1A, and the resulting protein-DNA complexes were resolved by native PAGE and visualized by PhosphorImager analysis. Lane NR (no receptor) represents an otherwise identical electrophoretic mobility shift experiment using extracts of Sf9 cells infected by a nonrecombinant baculovirus. The various RARα derivatives were tested either alone or together with the RXRα heterodimer partner, as indicated above the panels. Please note that 2–4-fold lower concentrations of receptor were used in the heterodimer assays compared with the homodimer assays (see “Materials and Methods”). The identities of these protein-DNA complexes were further probed by supershift experiment using RXRα (X) - or RARα (R) -specific antibodies, also as indicated above each panel (Ab). Representative electrophoretograms are presented; comparable results were obtained in triplicate experiments. A, DNA binding by receptor homodimers. B, DNA binding by heterodimers. Arrowheads point to presumed heterodimers of RXRα/RARα, RXRα/PML-RARα, and RXRα/PLZF-RARα.

Article Snippet: The electrophoretogram was then dried, visualized, and quantified by PhosphorImager analysis (Molecular Dynamics STORM system).

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Isolation, Infection, Incubation, Clear Native PAGE

Journal:

Article Title: DNA Recognition by the Aberrant Retinoic Acid Receptors Implicated in Human Acute Promyelocytic Leukemia 1

doi:

Figure Lengend Snippet: The DNA binding specificities of RARα, PML-RARα, and PLZF-RARα homodimers, as determined by gel electrophoretic mobility shift assay. A panel of radiolabeled oligonucleotide probes representing a series of single base substitutions relative to the consensus sequence were individually incubated with RARα (A), PML-RARα (B), or PLZF-RARα (C), and the resulting protein-DNA complexes were resolved by native PAGE and visualized by a PhosphorImager methodology. The nature of the base substitution in each oligonucleotide is indicated under the panel; in each case, the number refers to the position of the substituted base in the consensus half-site as described in the Fig. 1 legend, and the letter refers to the identity of the base substitution at that particular position (see “Materials and Methods” for further details of this nomenclature). Binding of each RARα derivative to the consensus sequence (CONS) was tested in Lane 1 of each panel. NR refers to negative controls using the consensus DNA element and nuclear extracts lacking retinoid receptors. Representative electrophoretograms are presented; comparable results were obtained in repeat experiments, which are quantified and presented in Fig. 4.

Article Snippet: The electrophoretogram was then dried, visualized, and quantified by PhosphorImager analysis (Molecular Dynamics STORM system).

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Sequencing, Incubation, Clear Native PAGE

Journal:

Article Title: DNA Recognition by the Aberrant Retinoic Acid Receptors Implicated in Human Acute Promyelocytic Leukemia 1

doi:

Figure Lengend Snippet: The half-site recognition properties of RXRα homodimers and a comparison of the DNA recognition properties of RXRα/RARα heterodimers relative to RARα homodimers. A, gel electrophoretic mobility shift assays, using our panel of consensus and nonconsensus half-site elements, were performed using RXRα. The protein-DNA complexes representing homodimers of RXRα were quantified for each half-site derivative by PhosphorImager methodology. Binding of RXRα to each probe is presented relative to the binding of the same protein to the consensus sequence (AGAGGTCA), which was set as 100%. The average obtained from two or more independent experiments is shown, as is the SD. B, the ability of RARα to bind to the different half-site DNA elements as a homodimer and as a heterodimer with RXRα (data in Fig. 4 and Fig. 8) was graphed as a difference plot (the binding of a given oligonucleotide probe by the heterodimer minus the binding of the same oligonucleotide probe by the analogous homodimer). Positive numbers indicate that the RXRα/RARα heterodimer binds the oligonucleotide better than does the RARα homodimer; negative numbers indicate that the homodimer binds better. The average obtained from multiple experiments, and the SD, are shown. Binding of homodimers and heterodimers to the different half-site sequences was defined relative to binding of the same receptor species to the consensus sequence; by this analysis, the difference between heterodimer and homodimer binding to the consensus sequences is defined as zero.

Article Snippet: The electrophoretogram was then dried, visualized, and quantified by PhosphorImager analysis (Molecular Dynamics STORM system).

Techniques: Comparison, Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing

Journal:

Article Title: DNA Recognition by the Aberrant Retinoic Acid Receptors Implicated in Human Acute Promyelocytic Leukemia 1

doi:

Figure Lengend Snippet: Half-site recognition properties of heterodimers of RXRα/RARα, RXRα/PML-RARα, or RXRα/PLZF-RARα. Gel electrophoretic mobility shift assays, using our panel of consensus and nonconsensus half-site elements, were performed as described in the Fig. 3 legend but in the presence of the heterodimer partner RXRα. The protein-DNA complexes representing heterodimers of RXRα/RARα (A), RXRα/PML-RARα (B), or RXRα/PLZF-RARα (C) were quantified for each half-site derivative by PhosphorImager methodology. Binding of each heterodimer to the different nonconsensus probes was normalized relative to the binding of the same heterodimer to the consensus sequence (AGAGGTCA), which was set as 100%. The average obtained from two or more independent experiments is shown, as is the SD.

Article Snippet: The electrophoretogram was then dried, visualized, and quantified by PhosphorImager analysis (Molecular Dynamics STORM system).

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing

Journal:

Article Title: DNA Recognition by the Aberrant Retinoic Acid Receptors Implicated in Human Acute Promyelocytic Leukemia 1

doi:

Figure Lengend Snippet: Comparison of DNA half-site recognition by retinoid receptor heterodimers versus homodimers: difference plots. A, EMSA analysis of DNA binding by PLZF-RARα homodimers and heterodimers. Preparations of RXRα and PLZF-RARα were mixed and tested for the ability to bind to the different response elements, as indicated. A PhosphorImager image of the resulting electrophoretogram is presented. Under these conditions, both PLZF-RARα homodimers (arrow) and RXRα/PLZF-RARα heterodimers (arrowhead) are observed. B, difference plot comparing RXRα/PLZF-RARα heterodimers with PLZF-RARα homodimers. The data in A was also quantified and graphed as a difference plot. Positive numbers indicate that the RXRα/PLZF-RARα heterodimer binds the oligonucleotide better than does the PLZF-RARα homodimer; negative numbers indicate that the homodimer binds better. The average obtained from multiple experiments and the SD are shown. C, difference plot comparing RXRα/ RARα heterodimers with PLZF-RARα homodimers. The ability of RXRα/RARα heterodimers to bind to the different DNA elements (Fig. 8A) was compared with that of PLZF-RARα homodimers (Fig. 4C). Positive numbers indicate that the RXRα/RARα heterodimer binds the oligonucleotide better than does the PLZF-RARα homodimer; negative numbers indicate that the homodimer binds better. The average obtained from multiple experiments and the SD are shown.

Article Snippet: The electrophoretogram was then dried, visualized, and quantified by PhosphorImager analysis (Molecular Dynamics STORM system).

Techniques: Comparison, Binding Assay