Skip to main content
. 2022 Feb 10;11(4):617. doi: 10.3390/cells11040617

Figure 6.

Figure 6

Impact of MRLC depletion on ABCB4 membrane stability. (A) Control HEK-293 cells (Ctrl) or HEK-CRISPR for MRLC (KO-MRLC) were transfected with ABCB4. After 24 h, cycloheximide (25 mg/mL) was added to the culture medium to inhibit protein synthesis. Expression of ABCB4 was analyzed by immunoblotting at the indicated time points, using equal amounts per line. a-Tubulin served as a loading control. Presented data were cropped from the full immunoblots shown in Supplementary Figure S6. (B) Amounts of ABCB4 were quantified from chase experiments. The amount of ABCB4 at time zero was considered to be 100%. The remaining ABCB4 at later time points was expressed as percentage of time zero. Means (±SD) of three independent experiments are shown. *** p < 0.001; ** p < 0.01; * p < 0.05. (C) MRLC is involved in the regulation of ABCB4 cell surface expression. HEK-293 cells-CRISPR for MRLC (KO-MRLC) stably expressing 3xmyc-ABCB4 were transiently transfected with a plasmid encoding MRLC-GFP. They were then incubated for 60 min at 0 °C with anti-myc antibody. After surface labeling, cells were fixed and ABCB4 was visualized with Alexa-Fluor 594-conjugated secondary antibody and visualized by confocal microscopy. Arrows point to MRLC knockout-3xmyc-ABCB4 expressing cells transfected with MRLC-GFP and arrowheads point to MRLC knockout-3xmyc-ABCB4 expressing cells that are not transfected with MRLC-GFP. Bars: 10 mm. (D) The amount of ABCB4 at the plasma membrane was quantified in MRLC knockout cells transfected with MRLC-GFP (KO-MRLC/MRLC-GFP) and compared to adjacent non-transfected cells expressing 3xmyc-ABCB4 (KO-MRLC) using ImageJ 1.41 software. Means (±SD) of at least 50 cells in two independent experiments are shown. **** p < 0.0001. (E) Impact of MRLC depletion on ABCB4 function. Control HEK-293 cells (Ctrl) or HEK-CRISPR for MRLC (KO-MRLC) were transfected with a plasmid encoding ABCB4, and PC secretion was measured after 24 h. Results are expressed as a percentage of PC secreted by ABCB4-transfected control cells with normalization to the amount of the mature ABCB4. Means (±SD) of at least two independent experiments performed in triplicate are shown. * p < 0.05.