Ird800 conjugated secondary antibodies

Cells were lysed in protein extraction buffer (Thermo Fisher, USA) on ice for 30 min. After centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatants were collected. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher, USA). For each sample, 20 μg total protein was resolved by SDS-PAGE and transferred to membranes to detect STAT3 (Cell Signaling Technologies, USA) and GAPDH (Cell Signaling Technologies, USA). After blocking in 5% milk powder, membranes were incubated with the primary antibodies at 4 °C overnight, washed, and then incubated with IRD800-conjugated secondary antibodies (Li-COR Biosciences, USA) at room temperature for 1 h in the dark. The membranes were then washed and analyzed using the Odyssey software system (Li-COR). STAT3 levels were normalized to GAPDH.