Journal: The Journal of Cell Biology
Article Title: 53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration
Figure Lengend Snippet: Loss of TP53BP1, USP28, or TRIM37 suppresses p53 elevation and proliferation arrest triggered by centrosome loss. (A, top) Outline of the procedure used to generate RPE1 knockouts. (A, bottom) Immunoblots of extracts from control (Ctrl) and knockout RPE1 lines. Bands corresponding to each protein (arrowheads) and nonspecific bands (asterisks) are indicated. α-Tubulin serves as a loading control. (B) Outline of cell proliferation analysis and assessment of p53 and p21 levels after acute treatment with centrinone or Mdm2i. (C) Graphs plotting the results of passaging assays monitoring the growth of control and knockout RPE1 cell lines after addition at day 0 of DMSO (vehicle), centrinone, or Mdm2i. (D) Immunoblots probed with the indicated antibodies after addition of Mdm2i (left) or centrinone (right). α-Tubulin (α-tub) serves as a loading control. (E) Immunofluorescence analysis of Cep192 and p53 after 5-d centrinone treatment. Representative images (left) and graph (right) plotting the distributions of nuclear p53 fluorescence for one of three experiments (for quantification of the other two experiments, see Fig. S2 E ). Graph shows 5–95% box-and-whiskers plots. Bar, 10 µm.
Article Snippet: To knock down TP53 (p53), RPE-1 cells were infected with a lentivirus containing sh-p53 made using the plasmid shp53 pLKO.1 puro (19119; Addgene; ).
Techniques: Western Blot, Knock-Out, Passaging, Immunofluorescence, Fluorescence