Ab155933

Cells were lysed in radio-immunoprecipitation assay buffer (ThermoFisher) with protease inhibitors (Roche, Basel, Switzerland). Samples were subjected to denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Gels were blotted on nitrocellulose membranes (GE Healthcare). After blocking with buffer (Li-Cor; Lincoln, NE, USA), the membrane was incubated with the following primary antibodies overnight at 4°C (S4 Table): mouse anti-STAT1 (ab155933), mouse anti-pSTAT1 (ab29045), mouse anti-STAT2 (sc-1668), rabbit anti-pSTAT2 (ab53132), mouse anti-STAT3 (ab119352), rabbit anti-pSTAT3 (ab76315), mouse anti-STAT5 (sc-74442), rabbit anti-pSTAT5 (ab32364), and mouse anti-β-Actin (ab8226) [Abcam, Cambridge, UK; Santa Cruz Biotechnology, Santa Cruz, CA, USA]. Next, the membrane was incubated with the appropriate IRDye goat anti-mouse and IRDye goat anti-rabbit secondary antibodies (Li-Cor). Prior to western blot analysis with the Odyssey Infrared Imaging System (Li-Cor), the combined linear range of detection for the targets and β-actin were determined. All images were analyzed using the Odyssey Application Software within the pre-determined ranges.

The PTC tissues and cells lysates were collected using RIPA buffer (Invitrogen, shanghai, China) and protease inhibitor cocktail (#5871; Beverly, MA, USA). Protein concentrations were calculated using Bicinchoninic Acid Kit (Takara; Merck KGaA) referring to the manufacturer’s protocols. Immunoblotting procedures followed the previously published method [13 ]. Equal amounts (60 µg) of total tissues or cellular proteins were added into loading buffer, boiled for 5 min, and separated on 10 % SDS-PAGE electrophoresis. Subsequently, the proteins were metastasized to nitrocellulose (NC) membranes. Then we blocked the membranes with 5 % non-fat dry milk for 1 h at room temperature and hatched overnight with primary antibodies. Following further incubated with secondary antibody for 1 h, the immunoreactive protein strips were visualized by ECL kit (Thermo, CA, USA) on the Bio-Rad Chemical Doc XRS system (Bio-Rad). The primary antibodies were including anti-p-p38 (ab195049; 1:2000), anti-p38 (ab250612; 1:1000), anti‐p-JAK2 (ab32101; 1:1500), anti‐JAK2 (ab39636; 1:800), anti‐p-STAT1 (ab215820; 1:1000), anti‐STAT1 (ab155933; 1:1000) and anti‐β‐actin (ab179467; 1:5000) were purchased from Abcam.