RPPA analysis identifies alterations of the cellular signaling network upon siRNA-mediated depletion of the novel hepcidin activators. Huh7 cells were transfected with the indicated siRNAs and protein extracts were collected 24 or 48 hours later. RNAi of HJV and BMPR1A was used as a positive control. Equal protein amounts were analyzed by RPPAs using antibodies that recognize total levels and/or phosphorylation abundance of 82 proteins involved in different signaling pathways. Because proteins involved in PI3K/Akt/mTOR and Ras/MAPK signaling are highly represented on the arrays, we further distinguished those signals that could serve as markers for the pathway activation and/or whose altered levels may exert their activation or suppression (supplemental Figure 5). Protein signals that were significantly altered compared with scrambled siRNA transfection are marked by a color code. Clustering of the obtained signal signatures at the 48-hour time point divides the analyzed hepcidin regulators into 2 groups, with either stronger (pink) or minor (green) effects on cellular signaling. On the right side, we further indicated how many protein signals (%) from each category were increased upon overall depletion of this group of hepcidin modifiers (pink) that confer strong alteration of the signaling signature.