tp53 d01 antibody  (Santa Cruz Biotechnology)

Journal: ACS pharmacology & translational science

Article Title: Covalent Modification of p53 by ( E )-1-(4-Methylpiperazin-1-yl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one.

doi: 10.1021/acsptsci.4c00447

Figure Lengend Snippet: Figure 1. NSC59984 reacts with free thiols via a Michael addition to the α-carbon. (A) Chemical structures of p53-reactivating molecules (NSC59984 and 1), MESNA (Sodium 2-mercaptoethanesulfonate (2)), adduct of Glutathione with NSC59984 (3), adduct of N-acetylcysteine with NSC59984 (4), and adduct of MESNA with NSC59984 (5). Potential sites for nucleophilic attack are labeled as α and β on the NSC59984 structure. The * label in 3, 4, and 5 represents the formation of a new stereogenic center after nucleophilic attack at the α carbon of NSC59984. Molecules 3 and 4 are a mixture of diastereomers, and 5 is a mixture of enantiomers. (B) Proposed scheme describing the reaction between 1 and 2. The * label represents the formation of a new stereogenic center after nucleophilic attack at the α carbon of 1. (C) 1H NMR analysis of time- dependent reaction of model compounds 1 and 2, showing thiol modification. Amplitude was adjusted to reduce signal-to-noise ratio in the stack plot so that specific peaks are more visible. (D) Quantum mechanical calculations of the LUMO for 1 (upper) or NSC59984 (lower) explain the pattern of reactivity.

Article Snippet: TP53 antibody (D01) (Santa Cruz) was diluted 1:50 before incubation, and a secondary antimouse AF647 antibody was used at 5 μg/mL before counter-staining.

Techniques: Labeling, Modification

Journal: ACS pharmacology & translational science

Article Title: Covalent Modification of p53 by ( E )-1-(4-Methylpiperazin-1-yl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one.

doi: 10.1021/acsptsci.4c00447

Figure Lengend Snippet: Figure 3. Modeling of potential structural effects of NSC59984 modification of p53 R248W. (A) Interactions of the Cys124-adduct with the loop of the L1/S3 pocket. In both panels the protein is represented as gray ribbon; side chains of Lys120 and Cys124 are shown as sticks; nitrogen, oxygen, sulfur and hydrogen atoms are colored blue, red, yellow and white, respectively; the carbons of the Cys124-adduct are colored magenta, and the carbons of Lys120 are colored cyan. (B) Cys124 and Cys229 adducts at the parallel interface of p53DBD monomers. The figure was made by superimposing trajectory snapshots onto the C (blue) and A (gray) subunits of a crystal structure of the p53 tetramer bound to DNA.22 The Cys229 and Cys124 adducts, carbons colored purple and magenta, respectively, are part of the top protein monomer. Panel (B-b) shows the Cys229-adduct making two possible hydrogen bonds represented by yellow lines. Panel (B-c) shows the Cys124-adduct making three possible, stabilizing interactions with the loop immediately following S4. The formal +1 charged nitrogen and the partially negatively charged hydroxyl oxygen of Ser166 are indicated by plus and minus signs.

Article Snippet: TP53 antibody (D01) (Santa Cruz) was diluted 1:50 before incubation, and a secondary antimouse AF647 antibody was used at 5 μg/mL before counter-staining.

Techniques: Modification

Journal: ACS pharmacology & translational science

Article Title: Covalent Modification of p53 by ( E )-1-(4-Methylpiperazin-1-yl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one.

doi: 10.1021/acsptsci.4c00447

Figure Lengend Snippet: Figure 4. Effects of biotinylated NSC59984. (A) Analysis of cellular proliferation was determined by fold change in CyQUANT measurement of cellular DNA following treatment with NSC59984-biotin (12 μM) for 72 h over carrier control in CP-A-WT or ESO26-R248W cells. Fold change in carrier-treated cells was normalized to 1. Statistical analysis was performed using a 2-way ANOVA test with Tukey correction. (B) Analysis of cellular proliferation was determined as in (A) following treatment with NSC59984 or NSC59984-biotin (12 μM) for 72 h over carrier control in ESO26-R248W cells. Fold change in carrier-treated cells was normalized to 1. Statistical analysis was performed using a 2-way ANOVA test with Tukey correction. (C) Western blot analysis of p53-biotin protein levels. CP-A-WT or ESO26 cells were treated with NSC59984-biotin (12 μM) for 72 h before cell lysis. Total protein was immunoprecipitated with Streptavidin Mag Sepharose beads and blotted for p53. (D) ESO26-R248W cells were treated with NSC59984-biotin (12 μM) for 2 h. Fixed cells were stained with streptavidin-AF488 (green), and counter stained for DNA (Hoechst, Blue). Images were captured on a Zeiss LSM710 confocal microscope. (E) ESO26-R248W cells were treated with NSC59984 (12 μM) for 12 h as described in panel (D).

Article Snippet: TP53 antibody (D01) (Santa Cruz) was diluted 1:50 before incubation, and a secondary antimouse AF647 antibody was used at 5 μg/mL before counter-staining.

Techniques: CyQUANT Assay, Control, Western Blot, Lysis, Immunoprecipitation, Staining, Microscopy