Complete inhibitors cocktail

Total proteins were obtained from tissue lysates with the RNA/Protein purification Kit (NorBiotek Canada, Thorold, ON, Canada). Additionally, protein extract from cell line were obtained by the RIPA 1× buffer supplemented with Complete^® inhibitors cocktail (Roche Applied Science, Mannheim, Germany). Proteins were separated by 10% SDS-PAGE, blotted onto nitrocellulose membranes, and blocked. The membranes were then exposed to mouse anti-Phospho STAT6 (1:1000) or anti-Total-STAT6 (1:1000), anti-SNAI1, anti-ERCC1, or anti-phospho-ERK1/2 and anti-Total ERK 1/2 antibodies (all from Cell Signaling Technology, Danvers, MA, USA). The anti-β-actin monoclonal antibody (Santa Cruz Biotechnology, St. Cruz, CA, USA) was utilized to detect cell actin as the protein load control. Horseradish peroxidase (HRP)-tagged secondary anti-rabbit or anti-mouse antibodies (1:5000) (Jackson Immunoresearch, West Grove, PA, USA) were used for chemiluminescent detection with the ImmobilonTM Western Chemiluminescent HRP Substrate kit (Millipore, Burlington, MA, USA). Chemiluminescence signals were recorded on Alliance Q9 UVITEC imaging device (Cambridge, UK) and densitometric analyses were performed with ImageJ software.

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.

Brain samples were homogenized 2 × 30 s at room temperature in (10% vol/w) 10 mM Tris, 0.32 M sucrose, pH 7.4 containing complete inhibitors cocktail (Roche) using ready to use Precellys Lysing Kit (Bertin Technologies) in a Minilys apparatus. After lysis, the homogenates were collected, frozen in liquid nitrogen and then stored at −80°C until use. When needed, frozen aliquots were diluted vol/vol with RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, pH 8) stirred 30 min at 4°C and then centrifuged 10 min at 14 000g at 4°C. Supernatants were frozen in liquid nitrogen and then stored at −80°C until use.