## 2 Materials and Methods
### Statistical Analysis  
The data were evaluated using GraphPad Prism 6 software. For variance analysis, an ordinary one-way or two-way analysis of variance test was performed using [PERSON]'s or [PERSON]'s multiple comparison test or paired or unpaired \(t\)-test as indicated. Results were considered significant with \(p<0.05\) (\({}^{\circ}p<0.05\), \({}^{\circ\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\! \!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\!\  
affinity pull-down (n = 3) and MALDI-TOF mass spectrometry using total cell lysates and purified GST-_hs_Nex, GST-_rt_Nex and GST, respectively. Pulldown samples were run on SDS-PAGE and stained with Coomassieie brilliant blue. Gel pieces were further processed and analyzed by mass spectrometry as described in Materials and Methods (Figure 1B, white boxes). The bands corresponding to GST-_hs_Nex, GST-_rt_Nex and GST were excluded from the analysis (Figure 1B, black boxes). All proteins interacting with GST-_hs_Nex, GST-_rt_Nex were detected and validated individually with a high degree of confidence based on the peptide sequences using specific databases and programs as described in Materials and Methods. The criteria for considering proteins being significant interactors of ERas Nex included their presence in all three independent pull-down experiments, their absence in the GST pull-down controls, removal of contaminant proteins arising from sample handling (such as keratin and bovine serum albumin). Collectively, we shortlisted a set of 76 ERas Nex potential interacting proteins, 35 of them were associated with _rn_Nex, 21 with _hs_Nex, and 20 proteins were found to bind to both Nex peptides (Figure 1C; Supplementary Table S1). Figure 1: ERas N-terminal extension and its novel binding partners. (**A**) ERas contains, in addition to five motifs (G1-5) in its G domain, an N-terminal extension (Nex) and a C-terminal hypervariable region (HVR) ending with a consensus sequence known as CAAX. An alignment of ERas N-terminus of _Rattus norregicus (rt)_ and _Homo sapiens_ (ls) shows a sequence identity (bold amino acids) of 42%. (**B**) Purified GST, GST-_rn_Nex or GST-_ls_Nex proteins were used for affinity pull-down experiments with GSH beads in total cell lysates, to identify ERas Nex binding partners. Bound proteins were resolved on a 10% SDS gel and stained with Coomassieie brilliant blue. White boxes indicate different gel fragments excised for mass spectrometric (MS) analysis. Black boxes indicate GST, GST-_rn_Nex or GST-_hs_Nex and were excluded from MS analysis (n = 3). (**C**). Evaluation of MS analysis revealed in total 76 ERas Nex binding partners from which 35 preferentially interact with _rn_Nex (yellow), 21 with _hs_Nex (red) and 20 with both (blue) (for more detail see Table S1). (**D**) Gene Ontology analysis of identified ERas Nex interacting proteins categorized according to biological processes, molecular functions, and subcellular localizations. Biological processes (left panel) were predominantly classified into metabolic pathways (29%), cell cycle control (24%), and cellular components and organization (16%). Molecular functions (middle panel) included: nucleic acid (RNA/DNA) binding proteins (27%), catalytic activity (18%) and protein binding (16%). Cellular components comprised predominated the cytosolic fraction (43%). (**E**) mRNA expression data of ERas Nex binding partners: arginase 1, nucleophosmin, lamin B1, and vimentin on day 0, 1, 4, and 8 of HSC cultivation (n = 3). The error bars indicate S.D. Identified proteins that interact with ERas Nex were classified into three ontologies: molecular function, biological process, and cellular component (Figure 1D). The vast majority of these proteins are involved in nucleic acid binding, molecular and catalytic activities and protein interactions. They are involved in the control of metabolic processes, cell cycle, and cell communication by localizing in different subcellular compartments, particularly in the cytosol.