Hela lysate

The slides were initially blocked with 1x Superblock® (Pierce, MA, USA) for 1 hour at 30°C. In vitro transcription translation (IVTT) was performed using HeLa lysate, Accessory Proteins, and Reaction Mix from Thermo Scientific (Carlsbad, CA, USA). Per the manufacturer’s recommendations, HeLa lysate was slowly injected onto the slide and the expression of the proteins was automated in two steps: 1. Hybridization at 30°C for 90 minutes with no agitation; 2. Hybridization at 15°C for 30 minutes with no agitation using an EchoTherm Chilling Incubator (Torrey Pines Scientific, Inc. Carlsbad, CA, USA). Following the IVTT step the slides are washed with 0.2% PBST then with with 5% milk in 0.2% PBST [21 (link)].

WT p53 full-length and other modified constructs were expressed as described below using Hela lysate (Thermo Scientific, Rockford, IL). After expression 5 μL of the recombinant protein was run on a 10% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was then probed with anti-GST MAb (Cell Signaling Technology, Danvers, MA), anti-p53 MAb DO.1, DO.14, and p240 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:1000 dilutions and signal was developed using Supersignal West Pico Chemiluminiscent substrate (Pierce, Rockford, IL, USA).