Chemidoc imaging device

Protein extracts were obtained from cell lysates with radioimmunoprecipitation assay (RIPA) 1× buffer supplemented with Complete^® inhibitors cocktail (Roche Applied Science, Mannheim, Germany). Proteins were separated by 10% SDS-PAGE and blotted onto nitrocellulose membranes and blocked. The membranes were then exposed to mouse anti-human TP63 (1:1500) or WIP1 (1:1000) antibodies (both from GeneTex, Irvine, CA, USA), anti-EGFR (1:1000), anti-p-Try1068-EGFR (1:1000), or anti-ATM (1:1000) antibodies (all from Cell Signaling Technology, Danvers, MA, USA). The anti-β-actin monoclonal antibody, kindly donated by Dr. JM Hernández (CINVESTAV-IPN), was utilized to detect cell actin as the protein load control. Horseradish peroxidase (HRP)-tagged secondary antibodies anti-rabbit or anti-mouse (1:5000) (Jackson Immunoresearch, West Grove, PA, USA) were used for chemiluminescent detection with the Immobilon™ Western Chemiluminescent HRP Substrate kit (Millipore, MA, USA). Chemiluminescence signals were recorded on a ChemiDoc imaging device (Bio-Rad Laboratories, Hercules, CA, USA) and densitometric analyses were performed with ImageJ software.

Protein extracts were obtained from cell lysates using 1× RIPA buffer supplemented with Complete^™ Protease Inhibitor Cocktail (Roche Applied Science, Mannheim, Germany). Protein concentrations were determined by the BCA method (Pierce™ BCA Protein Assay Kit). Thirty micrograms of protein were loaded per lane and separated by SDS-PAGE in 10% polyacrylamide gels, blotted onto nitrocellulose membranes and blocked with non-fat milk. The membranes were exposed to the anti-human antibodies listed in the Materials Section. The anti-actin monoclonal antibody, kindly donated by JM Hernández (CINVESTAV-IPN), was utilized to detect actin. The HRP-tagged secondary antibodies were anti-rabbit or anti-mouse (1:5000) (Jackson Immunoresearch, West Grove, PA, USA). Chemiluminescent detection was done with Immobilon^™ and recorded on a ChemiDoc imaging device (Bio-Rad Laboratories, Hercules, CA, USA) for densitometric analyses with ImageLab™ software (v 6.0, Bio-Rad Laboratories, CA, USA). All proteins were identified by Western blot from three independent experiments (biological replicates, n = 3).

5xFAD animals were bred in the Laboratory animal center of the University of Eastern Finland under the license approved by Regional State Administrative Agency of Finland (ESAVI‐2021‐002938) and Monash Animal Ethics Committee (MARP/2016/112) and conformed to national and institutional guidelines. In this study, a total of nine wild‐type and nine 5xFAD transgenic animals were used at the age of 5 and 5.5 months. Animals were housed in individually ventilated cages with unlimited food and water supply and 12 h light‐dark cycle. The animals were genotyped from ear samples. DNA was extracted by heating the samples to 95°C in 50 mM NaOH. pH was set after heating using 1 M Tris‐HCl, pH 8.0. Genotyping was done using primers for Psen1 (Integrated DNA Technologies, Inc., US) and DreamTaq Green DNA Polymerase (ThermoFisher Scientific, US). After polymerase chain reaction (PCR), the samples were run on agarose gel and imaged using ChemiDoc imaging device (BioRad Laboratories, Inc., US).