Tris buffered saline (tbs)

Protein extracts (30 µg/lane) were electrophoresed and electroblotted as previously described [18 (link)]. Electroblotted membranes were blocked with 5% non-fat milk (Nestlé Carnation, Solon, OH, USA) in TBS (Cell Signaling Technology, Boston, MA, USA) for 60 min at room temperature. They were then incubated with either primary 1:1000 antiphospho-Chk1[S345] (Cell Signaling Technology, #2348), 1:1000 anti-Chk1 (Cell Signaling Technology; #2360), 1:1000 anti-phospho-Chk2[T68] (Cell Signaling Technology; #2197), 1:1000 anti-Chk2 (Cell Signaling Technology; #6334), 1:500 anti-mutant p53 (abcam, Boston, MA, USA; #ab32049), 1:1000 anti-p53 (Santa Cruz Biotechnology, Dallas, TX, USA; #sc-6243), or 1:5000 anti-β-actin (Santa Cruz Biotechnology; #sc-47778) overnight at 4°C, followed by the corresponding horseradish peroxidase-conjugated goat anti-rabbit (Cell Signaling Technology; #7074) or anti-mouse (Cell Signaling Technology; #7076) secondary IgG for 2 h at room temperature in TBST (Cell Signaling Technology) with 5% non-fat milk (Nestlé Carnation) or 5% BSA (Cell Signaling Technology), according to the antibody manufacturer’s recommendations. Washed membranes were developed for optical density quantitation of chemifluorescent signals from replicate immunoblots as previously described [18 (link),24 (link)].

Protein lysates in 1X Laemmli buffer (Sigma-Aldrich) were separated on an SDS-10% polyacrylamide gel (Sigma-Aldrich) and the proteins were transferred onto nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). The membranes were blocked in TBS (Fisher Scientific, Loughborough, UK) containing 5% dried milk powder (w/v) and 0.1% Tween-20 (Fisher Scientific), for 1 h at room temperature. Following 3 washes with TBS-0.1% Tween-20, the nitrocellulose membranes were incubated with primary antibodies against Aβ⁴² and GAPDH (both from Cell Signalling Technology, Danvers, MA, USA). The primary antisera were used at a 1:1,000 dilution overnight at 4°C. The membranes were washed thoroughly for 30 min with TBS-0.1% Tween-20 prior to incubation with the secondary antibody, HRP-conjugated immunoglobulin (1:2,000; Sigma-Aldrich), for 1 h at room temperature and further washing for 30 min with TBS-0.1% Tween-20. Antibody complexes were visualised as previously described (21 (link)).

A431 ctr or KO cells were lysed on ice in NP-40 lysis buffer (100 mM NaCl 100 mM Tris pH8, 1% NP-40) for 15 min. Pellets were discarded after spinning at max speed for 15 min at 4 °C and supernatant was separated in SDS-PAGE and transferred onto nitrocellulose membrane. After blocking with 5% skimmed milk (Marvel, Camlab, Cambridge, UK) in TBS (Santa Cruz)/0.1% Tween20 (Sigma) for 30 min at RT, the membranes were incubated with primary antibodies in 5% skimmed milk/TBS-T. The following primary antibodies were used for western blotting: EGFR (Cell Signaling), pEGFR (Abcam), Actin (Millipore).The blots were washed three times with TBS-T and then incubated with corresponding IRDye secondary antibodies 680RD or 800CW (Li-Cor, Cambridge, UK, 0.05 µg/ml). After three times washing with TBS-T protein signals were visualized using the ODYSSEY Sa infrared system (Li-Cor) and Image Studio software 2.0 (Li-Cor). Full scans of Western blots are shown in Supplementary Figure 5.