Quantifying JAK1 and STAT3 Phosphorylation

Protein expression levels of p-JAK1 and p-STAT3 in the normal group, the TNFα group, and the TNFα + 10 μM DHC group were detected with ICF staining according to our previous study [3 (link)]. The samples were incubated with the primary antibodies anti-STAT3 (phospho Y705) (Abcam, Cambridge, MA, USA, 1:300) and anti-JAK1 (phospho Tyr1022) (Cell Signaling Technology, Beverly, MA, USA, 1:50) overnight at 4  °C. Then, the cells were incubated with FITC-conjugated secondary antibody (Beyotime, Shanghai, China, 1:300) for 1 h at room temperature (RT). Subsequently, cell nuclei were visualized with DAPI (Beyotime, Shanghai, China) staining for 15 min, and fluorescence images were obtained using fluorescent microscopy (Olympus, Tokyo, Japan). For each group, the fluorescence intensity was counted on five fields by Fiji-Image J analysis software (WS Rasband, National Institute of Health, Bethesda, MD, USA).