Direct enzyme-linked immunosorbent assay (ELISA) was performed to quantify the protein expression levels of cytoplasmic p-EGFR (Tyr 1092), and nuclear NF-κB (p65) and p-STAT3 (Tyr 705), in experimental and control treated-HCs, as previously described and in Supplementary Materials [90 (link)].
Briefly, BCA-200 Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect cytoplasmic and nuclear protein concentrations of all groups. Expression levels of p-EGFR, NF-κB, and p-STAT3 were then quantified by ELISA. The primary antibodies in our assay were: p-EGFR (Clone F-3), NF-κB(p65) (Clone F-6), and p-STAT3 (clone B-7) mouse monoclonal antibodies HRP (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Histone 1 (AE-4) and β-actin (Clone C4) (Santa Cruz Biotechnology Inc., Dallas, TX, USA) were used as a reference control for nuclear and cytoplasmic protein normalization, respectively). Absorbance values were measured by a microplate reader (Sunergy1, BIOTEK; Gen5TM software, BioTek Instruments Inc., Winooski, VT, USA). Assays were carried out according to the manufacturer’s instructions, performed in triplicates and repeated two times, independently.
Briefly, BCA-200 Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect cytoplasmic and nuclear protein concentrations of all groups. Expression levels of p-EGFR, NF-κB, and p-STAT3 were then quantified by ELISA. The primary antibodies in our assay were: p-EGFR (Clone F-3), NF-κB(p65) (Clone F-6), and p-STAT3 (clone B-7) mouse monoclonal antibodies HRP (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Histone 1 (AE-4) and β-actin (Clone C4) (Santa Cruz Biotechnology Inc., Dallas, TX, USA) were used as a reference control for nuclear and cytoplasmic protein normalization, respectively). Absorbance values were measured by a microplate reader (Sunergy1, BIOTEK; Gen5TM software, BioTek Instruments Inc., Winooski, VT, USA). Assays were carried out according to the manufacturer’s instructions, performed in triplicates and repeated two times, independently.
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