Chameleon duo

Untransfected control HEK293 cell lysate (30 μg) and HEK293 cell lysate overexpressing recombinant human C-MYC/FLAG-tagged THAP3 (30 μg)(Novus, #NBP2-05659) were separated by 4 – 12% BIS-Tris PAGE (Bio-Rad, #3450123). Chameleon Duo (7.5 μL) was used as the protein ladder (LI-COR, #928-60000). Proteins were separated for 1 hour at 180 V at room temperature. The protein transfer chamber was placed in an ice bucket, filled with 4°C transfer buffer (15% methanol and 85% 1x Tris/Glycine transfer buffer, Bio-Rad, #1610734), and proteins were transferred to Immun-Blot Low Fluorescence PVDF membrane at 100 V for 1 hour (Bio-Rad, #1620264). Membranes were blocked in undiluted blocking buffer (LI-COR, #927-70001) for 30 minutes at room temperature. The PVDF membrane was then incubated in CSF from Case 3 (1:60 in 1:1 LI-COR blocking buffer:TBST) overnight at room temperature. Membranes were rinsed five times in TBST and probed with goat-anti-human IRDye 800CW at 1:5,000 at room temperature for 1 hour. Membrane was rinsed five times in TBST and imaged on a LI-COR Odyssey. The same membrane was then probed with rabbit anti-FLAG antibody at 1:1,000 (Cell Signal, #2368) for 1 hour at room temperature, rinsed five times in TBST, probed with goat-anti-rabbit IgG at for 1 hour at room temperature, rinsed five times in TBST and reimaged on a LI-COR Odyssey.

To prepare a substrate for purified human 26S proteasome, p53 was polyubiquitinated with a MDM2 Ubiquitin Ligase Kit–p53 Substrate from Boston Biochem Inc. (Cambridge, MA), under conditions recommended by the manufacturer. After 15 min of polyubiquitination, 26S proteasome in buffer containing 1 μM TAT1-8,9TOD, TAT1-DEN, or vehicle was added to the reaction mixtures, and the reaction was carried out for 1 hour at 37°C. The reaction was stopped by the addition of SDS-PAGE buffer, as recommended. The samples were separated by SDS-PAGE (10% acrylamide tris-glycine precast Novex gels from Invitrogen, Carlsbad, CA), transferred to nitrocellulose membrane in a semidry system, and probed with anti-p53–specific monoclonal antibodies provided in the kit. Molecular weight standards Chameleon Duo (LI-COR Biosciences, Lincoln, NE) were run alongside reaction samples in each gel. Visualization of p53 was performed with an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) with secondary antibodies from Cell Signaling Technology (Danvers, MA).