FRAP analysis of vinculin mutants on polyacrylamide gels.

<p>(A) GFP-vinculin-expressing cells were cultured on soft (3.8 kPa) or rigid (25 kPa) substrates. FRAP analysis was performed and normalized fluorescence recovery of EGFP-vinculin was plotted using data from two independent experiments (n = 80). (B) The immobile fractions were calculated from fitted curves. The values represent the means ± S.E.M. Bonferroni’s test (n > 30; *P<0.05, **P<0.02; n.s., non-significant).</p

Actin co-sedimentation assay using vinculin mutants.

<p>(A) His-tagged WT vinculin (0.25 μM) with VBS3(talin) peptide (0, 10, or 25 μM) was incubated with F-actin, then ultracentrifuged. (B) His-tagged vinculin mutants (2 μM) and 25 μM VBS3(talin) were incubated with or without F-actin, then ultracentrifuged. (C) His-tagged vinculin mutants (1 μM) and 10 μM GST-VBS(IpaA) or GST were incubated with F-actin, then ultracentrifuged (right panel). Loading control (left panel). (D, E) The ratios of precipitated vinculin to total vinculin in B and C were quantified from three independent experiments using ImageJ. The asterisks indicate significant differences compared with WT vinculin using Bonferroni’s test (*P<0.05, **P<0.01).</p

Visualization and quantification of CSB-resistant vinculin mutants.

<p>(A) GFP-vinculin-expressing cells cultured on polyacrylamide gels were treated with CSB, then fixed and visualized using GFP. The insets indicate higher-magnification images of the areas indicated with dashed boxes. Scale bar: 20 μm. Fifty individual cells from three separate experiments were photographed for each condition. (B) FA numbers were quantified from (A) using ImageJ. The values represent the means ± S.E.M. One-way ANOVA, Scheffe’s test (n = 50; *P<0.05, ***P<0.001 compared with 3.8 kPa gel; n.s., non-significant).</p

Position 834 in TM6 plays an important role in cholesterol and phosphatidylcholine transport by ABCA1

<div><p>ATP-binding cassette protein A1 (ABCA1) plays a key role in eliminating excess cholesterol from peripheral cells by generating nascent high-density lipoprotein (HDL). However, it remains unclear whether both phospholipids and cholesterol are directly loaded onto apolipoprotein A-I (apoA-I) by ABCA1. To identify the amino acid residues of ABCA1 involved in substrate recognition and transport, we applied arginine scan mutagenesis to residues L821–E843 of human ABCA1 and predicted the environment to which each residue is exposed. The relative surface expression of each mutant suggested that residues L821–E843 pass through the plasma membrane as TM6, and the four residues (S826, F830, L834, and V837) of TM6 are exposed to the hydrophilic internal cavity of ABCA1. Furthermore, we showed that L834 is critical for the function of ABCA1.</p></div

Rescue of mislocalized NPC1L1^{L1072T/L1168I}.

<p>A, HEK293 cells stably expressing hNPC1L1-EGFP (WT) or hNPC1L1^{L1072T/L1168I}-EGFP (L1072T/L1168I) were incubated in the absence or presence of 10 µM fomiroid A for 24 h, and then plasma membranes (PM) were stained with CellMask Orange. The arrows show the rescue of mislocalized L1072T/L1168I mutant by fomiroid A treatment. Scale bar, 10 µm. B, WT or L1072T/L1168I mutant cells were treated with the indicated concentrations of fomiroid A for 24 h. The amount of NPC1L1 was determined using anti-GFP antibody. Vinculin was used as a loading control. C, Intensities of bands representing mature NPC1L1. Values represent means ± S.E. (n = 3). *<i>p</i><0.05 compared with untreated mutant cells.</p

Quantitation of cell-surface expression of HA-tagged NPC1L1^{L1072T/L1168I} by FACS analysis.

<p>HEK293 cells stably expressing HA-hNPC1L1^{L1072T/L1168I}-EGFP (HA-L1072T/L1168I) were incubated in the absence or presence of 10 µM fomiroid A for 24 h. Cell-surface expression of HA-L1072T/L1168I mutant was detected by staining with anti-HA and Alexa Fluor 633–conjugated anti–mouse IgG antibodies, and then quantitated by FACS analysis. The percentages in the upper right regions of the panels indicate the proportion of cells double-positive for anti-HA antibody and EGFP.</p

Structure of fomiroid A.

<p>Structure of fomiroid A.</p

¹H (500 MHz) and ¹³C (125 MHz) NMR data.

a, b<p>interchangeable.</p><p>¹H (500 MHz) and ¹³C (125 MHz) NMR data.</p

Dlg5 depletion promotes JNK and p38 activation.

<p><b>A:</b> LLc-PK1 cells were stimulated with 4 ng/ml TGF-β and incubated for the indicated periods. Cells were then lysed and immunoblotted using the indicated antibodies. <b>B, C, D, E:</b> LLc-PK1 cells were transfected with Dlg5 siRNA (KD) or control siRNA and incubated for two days. Cell lysates were then immunoblotted using the indicated antibodies, and the results were quantitated. The values represent the mean ± S.E. from at least three independent experiments. Dlg5 depletion promoted JNK and p38 activation but not Smad2/3 activation.</p

Dlg5 depletion disrupts epithelial cell morphology and induces the expression of mesenchymal marker proteins.

<p><b>A:</b> LLc-PK1 cells were transfected with Dlg5 siRNA (siDlg5#1: KD) or control siRNA and then incubated with 4 ng/ml of TGF-β for three days. The cells were lysed and immunoblotted using the indicated antibodies. Expression of β-tubulin was examined as a loading control. The upper band observed in immunoblotting with anti-Dlg5 is the larger variant of Dlg5. <b>B:</b> Cells were treated as in A and incubated for 60 hours. The cells were then photographed using phase contrast microscopy to examine cell morphology. <b>C:</b> Cells were transfected with Dlg5 siRNA or control siRNA and then immunostained using anti-Dlg5 or anti-E-cadherin antibodies. The asterisks indicate the cells that were transfected with Dlg5 siRNA. The scale bar indicates 10 µm. <b>D:</b> LLc-PK1 cells were transfected with Dlg5 siRNA or control siRNA and incubated for 24 hours. A GFP expression plasmid was then transfected into the cells with FLAG-Dlg5 or control plasmids. After incubation for two days, the cells were immunostained using an anti-SMA antibody. Fifty GFP-expressing cells were randomly selected, and the fluorescent intensity of SMA staining was examined. The graph shows the ratio of cells with higher SMA expression than background. <b>E:</b> LLc-PK1 cells were transfected with FLAG-Dlg5 or control plasmid and incubated for six hours. The cells were further transfected with Dlg5 siRNA or control siRNA. After two days of incubation, the cells were lysed and immunoblotted using the indicated antibodies. Arrows indicate endogenously expressed Dlg5, and an arrow head indicates FLAG-Dlg5. Dlg5 depletion induced the expression of mesenchymal marker proteins, and the re-expression of Dlg5 suppressed it.</p