Rabbit anti human ereg

Western blots were performed using an SDS–PAGE electrophoresis system as described previously [19 (link)], employing rabbit anti-human EREG (Abcam), rabbit anti-human SMA (Abcam), rabbit anti-human E-cadherin (CST), rabbit anti-human N-cadherin (CST), rabbit anti-human vimentin (Proteintech), rabbit anti-human p-JAK2 (CST), rabbit anti-human JAK2 (CST), rabbit anti-human p-STAT3 (CST), mouse anti-human STAT3 (CST), rabbit anti-human p-NF-kB (CST), rabbit anti-human NF-kB (CST), rabbit anti-human p-p38 (CST), rabbit anti-human p38 (CST), rabbit anti-human p-akt (CST), rabbit anti-human akt (CST), rabbit anti-human p-erk1/2 (CST), rabbit anti-human erk1/2 (CST), rabbit anti-human snail (CST), antibodies. The blots were re-probed for rabbit anti-human GAPDH (Bioworld) to control for protein loading and transfer.

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.