Histone 1 ae 4

Western blot analysis and a direct enzyme‐linked immunosorbent assay (ELISA) were performed, as previously described⁷, ¹³, ¹⁶, ³¹ and in Supplementary Material, to monitor the successful knockdown of STAT3 expression, and to determine the effect of STAT3 knockdown or its pharmacologic inhibition on BA‐induced STAT3, NF‐κB and Bcl‐2 protein levels. We used primary antibodies for p‐STAT3 (Tyr 705) (clone B‐7), STAT3 (clone F‐2), p‐NF‐κB (p65 Antibody 27. Ser 536), bcl2 (Clone N‐19), Histone 1 (AE‐4) and β‐actin (C4) (Santa Cruz Biotechnology Inc.). Protein levels obtained by Western blot analysis were quantified by the Gel imaging system (Bio‐Rad) in each nuclear or cytoplasmic cellular compartment (Image Lab 5.2 analysis software, Bio‐Rad). Protein levels obtained by ELISA were quantified by Gen5™ software reading the absorbance values using a microplate reader (Sunergy1, BIOTEK; Gen5™ software; BIOTEK Instruments Inc.). Assays were carried out according to the manufacturer's instructions and performed in triplicates and repeated two times, independently.

Western blot analysis was used to determine the cytoplasmic or nuclear protein levels of p-EGFR (Tyr1092), bcl-2, p-NF-κB (p65 S536), and p-STAT3(Tyr705), in experimental and control treated-HCs. Beta-actin and Histone 1 were used to normalize cytoplasmic and nuclear extracts, respectively, as previously described and in Supplementary Material [56 (link),58 (link)]. We used 1:1000 primary antibodies for p-EGFR (Clone F-3), bcl2 (Clone N-19), p-NF-κB (p65 Antibody 27.Ser 536), p-STAT3 (Tyr 705) (clone B-7), Histone 1 (AE-4) and β-actin (C4) (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Protein levels were quantified by the Gel imaging system (Bio-Rad, Hercules, CA, USA) in each cytoplasmic or nuclear cellular compartment (Image Lab 5.2 analysis software, Bio-Rad, Hercules, CA, USA).

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; Gen5^TM 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.