rabbit polyclonal antibody against p53  (Proteintech)

Journal: Molecular cancer research : MCR

Article Title: The C-terminus of gain-of-function mutant p53 R273H is required for association with PARP1 and Poly-ADP-Ribose

doi: 10.1158/1541-7786.MCR-22-0133

Figure Lengend Snippet: (A) Schematic of mutant p53 R273H protein alterations as a result of CRISPR/Cas9 alteration or site-directed mutagenesis. (B) Western blot from whole cell lysates of MDA-MB-468 cells comparing expression levels of mtp53 R273H, mtp53 R273HΔ381–388, R273HΔ347–393, and mtp53 R273Hfs387. (C) Western blot from whole cell lysates of HCT116 p53−/− cells transfected with plasmids encoding p53 with the same deletions. Transfection with eGFP-expressing plasmid is used as a negative control for p53 expression.

Article Snippet: Rabbit polyclonal antibodies against p53 (Cat# 10442–1-AP) and PARP1 (Cat# 13371–1-AP) were purchased from Proteintech.

Techniques: Mutagenesis, CRISPR, Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control

Journal: Molecular cancer research : MCR

Article Title: The C-terminus of gain-of-function mutant p53 R273H is required for association with PARP1 and Poly-ADP-Ribose

doi: 10.1158/1541-7786.MCR-22-0133

Figure Lengend Snippet: (A) Analysis of p53/PARP1 complexes by in situ proximity ligation assay (PLA) in MDA-MB-468, R273HΔ381–388 and R273HΔ347–393 cells. Fluorescent foci per cell were counted by Cellprofiler software and depicted by a scatter plot using GraphPad Prism 9. The data represent a scatter plot with n=3. An ordinary one-way ANOVA was used to determine the statistical significance of the data. The following format was used to assign significance based on P-value: **** represents a p-value ≤ 0.0001 and ns represents non-significant. Representative confocal microscope images of p53/PARP1 foci/complexes (red) by in situ proximity ligation assay (PLA) in MDA-MB-468, R273HΔ381–388 and R273HΔ347–393 cells. DNA was counterstained with DAPI (blue). The z-stack maximum intensity projection images are shown. Three independent experiments were performed. (Scale bar = 10μm). The negative control scoring for both panels A and B was single antibody probing for p53 followed by all other PLA steps. (B) Analysis of p53/PAR complexes by in situ proximity ligation assay (PLA) in MDA-MB-468, R273HΔ381–388 and R273HΔ347–393 cells. Fluorescent foci per cell were counted by Cellprofiler software and depicted by a scatter plot using GraphPad Prism 9. The data represent a scatter plot with n=3. An ordinary one-way ANOVA was used to determine the statistical significance of the data. The following format was used to assign significance based on P-value: **** represents a p-value ≤ 0.0001 and ns represents non-significant. Representative confocal microscope images of p53/PAR foci/complexes (red) by in situ proximity ligation assay (PLA) in MDA-MB-468, R273HΔ381–388 and R273HΔ347–393 cells. DNA was counterstained with DAPI (blue). The z-stack maximum intensity projection images are shown. Three independent experiments were performed. (Scale bar = 10μm). (C) Pellets from MDA-MB-468, R273HΔ381–388 and R273HΔ347–393 cells were lysed in NP-40 buffer and lysate used for co-immunoprecipitation assay. Samples were run on an SDS-PAGE gel and western blot analysis performed with p53 and PARP1 antibodies. Lanes 1,4 and 7 represent the input lanes, lanes 2,5 and 8 represent the negative control IgG lanes and lanes 3, 6 and 9 represent the p53: IP lanes. Image is a representation of 3 independent biological replicates.

Article Snippet: Rabbit polyclonal antibodies against p53 (Cat# 10442–1-AP) and PARP1 (Cat# 13371–1-AP) were purchased from Proteintech.

Techniques: In Situ, Proximity Ligation Assay, Software, Microscopy, Negative Control, Co-Immunoprecipitation Assay, SDS Page, Western Blot