Supplementary Figure 4: IL-6 production by Kupffer cells after various stimuli and inhibitor treatments.

(a,b) ELISA quantifying IL-1β-induced IL-6 secretion by isolated Kupffer cells after preincubation with increasing amounts of the EGFR inhibitors Cetuximab (a) or BIBW2992 (Afatinib) (b). (n = 2 primary Kupffer cell isolates). (c) IL-6 secretion by isolated Kupffer cells following stimulation with polyIC (20 μg ml⁻¹), imiquimod (12 μg ml⁻¹) and LPS (10 ng ml⁻¹). - = unstimulated. (n = 2 primary Kupffer cell isolates). (d) ELISA quantifying IL-1β- or EGF-induced IL-17A, IL-22 and IL-23 secretion by isolated Kupffer cells (n = 2 primary Kupffer cell isolates). (e,f) Representative Western Blot showing activation of the indicated proteins after 15 min stimulation with IL-1β (e) or EGF (f) in the presence of the respective inhibitors. Note: Each lane contains proteins isolated from pooling Kupffer cells of 3 different livers. Because the amount of proteins obtained from Kupffer cells from 3 pooled livers was not sufficient to perform Western blot analysis for all indicated proteins and treatments, 2 different isolates and Western blots for each series of treatment (EGF+ inhibitors and IL-1β+ inhibitors) had to be performed. (e) Blot 1: IL-1β stimulated EGFR^f/f and EGFR^ΔMx+ inhibitors and expression of EGFR and JNK. Blot 2: IL-1β stimulated EGFR^f/f and EGFR^ΔMx+ inhibitors and expression of p38, IKK, NF-kB, Stat3. (f) Blot 1: EGF stimulated EGFR^f/f and EGFR^ΔMx+ inhibitors and expression of EGFR and JNK. Blot 2: EGF stimulated EGFR^f/f and EGFR^ΔMx+ inhibitors and expression of p38, IKK, NF-kB, Stat3. The results were confirmed in a second set of isolates and Westerns. Original data are provided in Supplementary Table 1.