G59 12 clone

Cells were incubated in serum-free medium for 4 h followed by irradiation (1.8 Gy). After 24h, cells were lysed and 50 µg of proteins from each sample were run on a 6–12% SDS-PAGE gel and transferred to a PVDF Hybond-P (GE Healthcare, Brazil) membrane. Membranes were incubated with antibodies against EGFR (1:500; #2232, Cell Signaling Technology, USA), ERCC1 (1:100; 8F1 clone, Thermo Fisher Scientific, USA), p53 (1:500; G59–12 clone, BD Pharmingen, USA) and HSC70 (heat shock cognate 70 protein; 1:5000; Santa Cruz Biotechnology, USA). After incubation with secondary antibodies, immunoblots were detected using the ECL reagent (GE Healthcare, Brazil). Predicted molecular weights for EGFR, ERCC1, p53, and HSC70 are 170, 39, 53, and 70 kDa, respectively.

For immunohistochemistry, TMA was constructed, as described by Pires et al. (19 (link)). All samples were fixed in 10% formalin and embedded in paraffin. Serial TMA sections were cut, mounted on glass slides and dried at 56°C before dewaxing in xylene and rehydration in alcohol. All sections were subjected to heat-induced epitope retrieval in citrate buffer followed by inhibition of endogenous peroxidase (peroxidase block, RE7101, Novocastra, UK). Incubation of primary antibodies against EGFR (D38B1, 1:25, Cell Signaling Technology), ERCC1 (1:100; 8F1 clone, Thermo Fisher Scientific), or p53 (1:100; G59–12 clone, BD Pharmingen) was performed for 1 h. Slides were incubated with NovoLink™ polymer (RE7112, Novocastra), further developed with diaminobenzidine chromogen (RE7105, Novocastra), and finally stained with Mayer hematoxylin, dehydrated and mounted with Canadian balsam. EGFR, ERCC1 and p53 expressions were evaluated semi-quantitatively as positive cells after counting 300–500 tumor cells, being scored as negative (no staining), 1+ (<25% of positive cells), 2+ (25–75% of positive cells) or 3+ (>75% cells staining positively). Negative controls were obtained by omitting the primary antibody.