## 2 Materials and Methods
### ERas-ARG1 Interaction in Quiescent HSCs  
In proteomic analysis, we identified ARG1 as a potential binding partner of _hs_Nex and _rn_Nex (Supplementary Table S1), which is also upregulated in quiescent HSCs. Isolated rat HSCs activate during culture on plastic surfaces. Here, they switch from a quiescent state (qHSC) to an activated state (aHSC) (Figure 2A) as they do in response to, for example, chronic liver injury. Day 0 HSCs are considered as quiescent cells, expressing the marker proteins GFAP and desmin, whereas day 8 HSCs are activated and display the activation marker \(\alpha\)-SMA. ERas was found to be exclusively expressed at day 0 and also ARG1 exhibited the highest expression at that day 0. The ARG1 expression decreases at day 1 and increases again at day 4 and 8 (Figure 2B). Interestingly, the inducible nitric oxide synthase (iNOS), which uses, like ARG1, L-arginine as substrate, was reciprocally expressed and could only be detected at day 1. Figure 2: ERas and ARG1 expression, localization, and interaction in hepatic stellate cells. (**A**) Graphic illustration of quiescent (qHSCs) and activated hepatic stellate cells (aHSCs). During culture, the HSCs change in their cell shape and size, and reduce their lipid droplet content. (**B**) Immunoblot analysis of ERas and ARG1 from freshly isolated (d0) and activated HSCs maintained in monoculture for up to 8 days (d1, d4, d8). GFAP was used as a marker for quiescent HSCs (d0), and \(\alpha\)-SMA was  
used as a marker for activated HSCs (d8). \(\gamma\)-tubulin served as a loading control. (**C**) Pull-down assay of ARG1 and GST-_rn_Nex in freshly isolated HSC (d0) lysates. Input and output were immunoblotted and detected with anti-ARG1 antibody. (**D**) Confocal imaging of GFAP, ARG1 and ERas of freshly isolated HSCs after 6 h of culture at day 0 shows colocalization of ERas and ARG1 (white arrows). Scale bar: 10 \(\upmu\)m. Six-fold magnification of the merged image. (**E**) Cell fractionation of HSCs d0 in five distinct fractions including: heavy membrane (HM; plasma membrane and rough endoplasmic reticulum), light membrane (LM; polysomes, Golgi apparatus, smooth endoplasmic reticulum), cytoplasm (CP; cytoplasm and lysosomes), nucleus (Nu) and total cell lysate (TCL). Immunoblot analysis was performed for Na\({}^{+}\)/K\({}^{+}\)-ATPase, Histone H3, Actin, GAPDH, ERas and ARG1. (**F**) Pull-down assay of purified ARG1 protein with GST-Nex or GST-FL of human and rat ERas. (**G**) Immunoprecipitation (IP) analysis of ARG1 with various ERas constructs overexpressed in HEK 293 cells (_hs_ERas FL, _rn_ERas FL, _hs_ERas Nex, _rn_ERas Nex, _hs_ERas\({}^{\Delta C}\), _rn_ERas\({}^{\Delta C}\), empty vector). IP was conducted using GFP-coupled nanobeads. Empty EYFP served as a control. Protein complexes retained on the beads were resolved by Laemmli buffer and processed for immunoblotting using a monoclonal antibody against ARG1. (**H**) Sensorgrams obtained from the binding of 0.5-20 \(\upmu\)M ARG1 to immobilized GST-_hs_Nex on the surface of a CM5 sensor chip. The interaction of ERas and ARG1 was verified by pull-down (Figure 2C) and IP experiments (Supplementary Figure S1), where ARG1 could be pulled down with purified GST-bound ERas _rn_Nex or coprecipitated by fishing with an ERas antibody in freshly isolated HSC d0 cell lysates, respectively. Next, confocal imaging revealed a strong colocalization of ERas and ARG1 in freshly isolated and 6 h cultivated HSCs (Figure 2D, white arrows, additional images in Supplementary Figure S2). The marker GFAP was stained as a quiescent HSC marker. The subcellular localization of ARG1 and ERas was further determined via cell fractionation (Figure 2E). Here, both proteins could be detected in heavy membrane (plasma membrane and rough endoplasmic reticulum), light membrane (polysomes, Golgi apparatus, smooth endoplasmic reticulum) and cytoplasmic fraction (cytoplasm and lysosomes). ARG1 could additionally be found in the nuclear fraction.