Immunoblotting Analysis of p53 Regulatory Proteins

Protein was extracted, quantified, and probed with antibodies, as described.^{40 (link)} Whole cell lysates were extracted using RIPA buffer (Cell Signaling). Antibodies used included p53 (Cell Signaling, Danvers, MA), Mdm2 (EMD Millipore, Billerica, MA), WIP1 (Bethyl, Montgomery, TX), and β-actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies Alexa Fluor 680 goat anti-mouse IgG (Life Technologies) or IRDye 800 goat anti-rabbit IgG (Rockland, Gilbertsville, PA) were used at a dilution of 1:5,000. Immunoblots were imaged as previously described.^{29 (link)}

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Wen J., Lee J., Malhotra A., Nahta R., Arnold A.R., Buss M.C., Brown B.D., Maier C., Kenney A.M., Remke M., Ramaswamy V., Taylor M.D, & Castellino R.C. (2016). WIP1 modulates responsiveness to Sonic Hedgehog signaling in neuronal precursor cells and medulloblastoma. Oncogene, 35(42), 5552-5564.

Publication 2016

RIPA buffer β-actin Alexa Fluor 680 goat anti-mouse IgG IRDye 800 goat anti-rabbit IgG

Actin Anti igg Antibodies Buffer Cell Dilution Goat Immunoblots Irdye 800 Mdm2 Mouse Protein Rabbit Ripa

Corresponding Organization :

Other organizations : Center for Cancer and Blood Disorders, Aflac (United States), Emory University, Hospital for Sick Children, SickKids Foundation, University of Toronto

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Variable analysis

independent variables

Protein extraction
Protein quantification
Probing with antibodies

dependent variables

Protein expression levels

control variables

RIPA buffer used for whole cell lysate extraction
Antibodies used: p53, Mdm2, WIP1, β-actin
Secondary antibodies used: Alexa Fluor 680 goat anti-mouse IgG, IRDye 800 goat anti-rabbit IgG

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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