*2.1. Identification of RAB10 as a New Molecular Partner of ABCB4*

Despite the important role played by ABCB4 in bile secretion, little is known about the molecular mechanisms regulating its expression, intracellular traffic, and function. To clarify some of these mechanisms, the aim of this study was to identify key players implicated in ABCB4 regulation. For this task, an AP-MS approach was used (see details in the Section 4). Among several ABCB4 candidate interactors (Supplementary Table S1), we found the small GTPases RAB10 and RAB13 of particular interest, as RAB11 has been involved in the traffic of the canalicular bile salt export pump ABCB11 [27]. Our preliminary results indicated less encouraging results for RAB13 (data not shown), so we decided to

further investigate the role of RAB10 in the intracellular traffic and function of ABCB4. To confirm the interaction between ABCB4 and RAB10, co-immunoprecipitation experiments were performed in primary human hepatocytes. RAB10 was detected after ABCB4-specific immunoprecipitation but not in the control condition with unspecific antibodies (Figure 1A). Conversely, ABCB4 was specifically detected from RAB10-immunoprecipitated complexes (Figure 1B). The same results were observed in HEK cells co-transfected with ABCB4 and RAB10-GFP (Supplementary Figure S1). These results confirm the interaction between ABCB4 and RAB10.

**Figure 1.** Co-immunoprecipitation of ABCB4 and RAB10 in primary human hepatocytes. (**A**,**B**) ABCB4 (A) or RAB10 (B) were immunoprecipitated from primary human hepatocyte lysates using specific antibodies. Controls were performed using unspecific antibodies (unspe.). After SDS-PAGE, the presence of ABCB4 and RAB10 in the lysates (Input) and the immunoprecipitates (IP) was detected by immunoblot (IB) as indicated. Molecular weight markers (in kDa) are indicated. These panels are representative of at least three independent experiments per condition. The presented data were cropped from full immunoblots shown in Supplementary Figure S2.
