Phospho jak2

Immunofluorescence of cryosections or immunohistochemistry of paraffin-embedded tissue was done as previously described [19 (link)]. Primary antibodies were rabbit anti-human EREG (Abcam), phospho-JAK2 (Proteintech), phospho-STAT3 (CST), SMA (Abcam).
EREG expression in CAFs was identified on the basis of SMA staining. CAFs were defined as SMA positive, spindled-shaped cells in the stroma (data of SMA staining not shown). EREG staining was assessed only in fibroblasts in tumor stroma by the evaluation of staining intensity and the percentage of EREG positive fibroblasts according to criteria modified from Zhang J et al. (Fig. 2a). Briefly, EREG expression was scored by a combination of staining intensity (score: 0 = no staining, 1 = weak staining, 2 = medium staining, 3 = strong staining) and percentage of EREG positive cells (score 0 = 0%~ 5% positive cells, 1 = 6%~ 33% positive cells, 2 = 34%~ 66% positive cells and 3 = 67%~ 100% positive cells) by a method modified from Zhang J, et al. (Fig. 2a) [1 (link)]. EREG staining index was taken as the product of staining intensity and percentage. The staining index was further divided into low and high: score 0–4 (negative to medium) was defined as EREG low, score 6 and 9 (strong) was defined as high EREG expression. Two observers examined the images independently without the knowledge of patients’ information.

The rat livers and AML12 cells were lysed by RIPA buffer, separated by 10% polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membrane. After blocking in 5% Bovine Serum Albumin (BSA), the membrane was incubated with the phospho-JAK2 (Tyr931) (1:1,000, affinity), total JAK2 (1:1,000, proteintech), phospho-STAT3 (Tyr705) (1:1,500, abcam), total STAT3 (1:2000, abcam), IL-6 (1:500, bioworld), IL-18 (1:2000, bioworld), IL-1β (1:500, sino biological), TNFα (1:1,000, sino biological), HIF-1a (1:1,000, Novusbio), HIF-2a (1:1,000, Cell Signaling Technology), VEGFa (1:1,000, Novusbio) primary antibody seperately overnight at 4°C, and then was incubated with HRP conjugated secondary antibody for 1 h at room temperature. Western blots were displayed by advanced enhanced chemiluminescence kit and then semi-quantified by Clinx (Shanghai Qinxiang Scientific Instrument Co., Ltd).