Electrochemiluminescence kit

After transfection for 48 h, cells in different treatment groups were washed with pre-cooled phosphate buffered saline (PBS; Thermo fisher, USA) for 3 times. Whole cell lysate was then used for cell lysis on ice for 10 min, and the extracted protein samples were quantitated using the BCA protein assay kit (Thermo Fisher Scientific, USA). Subsequently, the protein samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V following 10 min of boiling at 95 ℃ with 10 μl of loading buffer, after which the separated proteins were loaded on a nitrocellulose membrane at 100 mA within 120 min. After blocked with 5% bovine serum albumin (BSA) or Tris-Buffered Saline Tween (TBST) for 60 min, the membrane was incubated overnight at 4 °C with primary antibodies, followed by addition of secondary antibody goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) for hybridization at a room temperature within 120 min. The membrane was transferred to a shaking table for wash with 1 × TBST (Solarbio, Beijing, China) before and after the second incubation, with the wash respectively ran for 3 times. The electrochemiluminescence kit from Solarbio (Beijing, China) was employed to visualize protein bands. All antibodies used were detailed in Additional file 1: Table S2.

After 48 h of transfection, the cells were washed with pre-cooled PBS (Thermo Fisher Scientific, MA, USA) 3 times and then lysed in RIPA lysis buffer (Thermo Fisher Scientific, MA, USA) on ice for 10 min. The BCA assay kit from Thermo Fisher Scientific (MA, USA) was applied for quantification of the protein samples. After boiled at 95°C for 10 min, the protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 100V and then transferred to nitrocellulose membranes. Five percent BSA/TBST was used to block the membranes for 60 min. Subsequently, the membranes were incubated with primary rabbit polyclonal antibodies overnight at 4°C and with horseradish peroxidase (HRP) -conjugated secondary antibody goat anti-rabbit (ab6721, 1:3000, abcam, Cambridge, UK) in succession for 120 min of hybridization at room temperature. 1 × TBST (Solarbio, Beijing, China) was used to wash the membranes. Protein bands were visualized using the electrochemiluminescence kit (Solarbio, Beijing, China) and photographed. Primary antibodies include CXCL1 (PAI-29220, 1:2000, Thermo Fisher Scientific), Jak2 (ab108596, 1:5000, abcam), p-Jak2 (ab76293, 1:5000, abcam), STAT3 (9133S, 1:1000, Cell Signaling Technology), p-STAT3 (8204S, 1:1000, Cell Signaling Technology), and GAPDH (ab181602, 1:10000, abcam).