Western Blot Analysis of CXCL1 and NF-κB Signaling

As described before [17 (link)], aliquots of 40 μg of protein from each group was separated by 13% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a 0.22-μm polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in TBS + 0.05% Tween™ 20 for 1 hour at room temperature, membranes were incubated with an Rabbit Anti-CXCL1 antibody (Proteintech, Chicago, IL, USA), Rabbit Anti-Phospho-NF-κB p65 (Ser536) antibody, Rabbit Anti-NF-κB P65 antibody (Cell signaling Technology, Beverly, MA, USA), or Anti-GAPDH (Bioworld, Nanjing, China) overnight at 4°C. Densitometric levels of CXCL1 signals were quantified and expressed as their ratio to GAPDH.