*4.4. Immunoblots, Immunofluorescence and Measurement of ABCB4-Mediated Phosphatidylcholine Secretion*

Immunoblots were performed as previously described [9,45], using cells co-transfected with ABCB4- and RAB10-encoding constructs at a 1:1 ratio with the following primary

antibodies: anti-ABCB4 (clone P3II-26) from Enzo Life Sciences, anti-α-tubulin (clone 1E4C11) from ProteinTech (Manchester, UK), anti-RAB10 (clone D36C4) from Cell Signaling (Danvers, MA, USA) and anti-GFP (clone AB10145) from Sigma. Immunoblots were quantified in the linear range of detection using ImageJ 1.50i software (U.S. National Institutes of Health, Bethesda, MD, USA).

For indirect immunofluorescence experiments, cells were grown on glass coverslips, fixed with 4% paraformaldehyde (Thermo Fisher Scientific) for 10 min, and permeabilized with 0.05% saponin in PBS containing 0.2% of bovine serum albumin (BSA). The coverslips were then incubated with the following primary antibodies: anti-ABCB4 and anti-RAB10 (see above), anti-calnexin (SPA-865, Enzo Life Sciences), anti-giantin (PRB-114C, Covance, Rueil-Malmaison, France), anti-EEA1 (sc-6415, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-LAMP1 (sc-20011, Santa Cruz Biotechnology); and subsequently incubated for 1 hour with appropriate Alexa Fluor-conjugated secondary antibodies (ThermoFisher Scientific). Nuclei were labeled using Hoechst 33342 (ThermoFisher Scientific). Immunofluorescence images were acquired using a confocal microscope (Eclipse TE-2000-Nikon-C2) equipped with a 60X objective, serial xy optical sections with a z-step of 0.3 µm were acquired using Nikon NIS-Elements software version AR 4.50 with constant settings (laser powers and correction of signal intensities) and treated using Adobe Photoshop version 8.0.1.

Alternatively, for specific cell surface labeling of ABCB4, non-permeabilized HeLa cells expressing ABCB4-FLAG were incubated 1 h with polyclonal anti-FLAG antibodies (F7425, Sigma) prior to fixation. Then, after fixation and permeabilization, total vs. cell surface ABCB4 were revealed using the standard procedure described above. A cell by cell quantification of cell surface vs. total ABCB4 signal intensities was performed as follows: individual ABCB4-expressing cells were randomly selected and delineated in ImageJ software, version 1.50i using the total ABCB4 signal. For each segmented cell, the fluorescence intensities associated with total ABCB4 (shown in blue) or plasma membrane ABCB4 (shown in red) were measured in each channel using the 'Measure' function of ImageJ. Then, after background subtraction (measured in areas with no apparent fluorescence) for each fluorescence intensity, plasma membrane over total ABCB4 fluorescence ratios were expressed as percentages of the mean ratio calculated for the reference condition (control).

The measurement of ABCB4-mediated PC secretion using a fluoro-enzymatic assay was performed as described [49] and results were analyzed as published [45].
