Horseradish peroxidase hrp conjugated goat anti rabbit secondary antibodies

Proteins were extracted from lung tissues using RIPA buffer [Cell Signaling Technology (CST), USA], and the concentrations were quantified using a BCA protein assay reagent (Beyotime, Nanjing, China) according to the manufacturer's instructions. Equal amounts of protein extracts were resolved by 8 or 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were subsequently incubated with primary antibodies, including rabbit anti-mouse STAT3 polyclonal antibodies (1:1,000, CST, USA), rabbit anti-mouse p-STAT3 polyclonal antibodies (1:1,000, CST, USA), and rabbit anti-mouse GAPDH polyclonal antibodies (1:5,000, Proteintech, China) at 4°C overnight. The membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (1:5,000, goat, zsgb-bio, China) at room temperature for 1 h. The protein bands were visualized using the enhanced chemiluminescence method (GE Healthcare Life Sciences, Little chalfont, UK) and quantified by using Quantity One software (Bio-Rad, Hercules, CA). GAPDH was used as the internal reference.