Journal: Nature Communications

Article Title: PPFIA1 drives active α5β1 integrin recycling and controls fibronectin fibrillogenesis and vascular morphogenesis

doi: 10.1038/ncomms13546

Figure Lengend Snippet: ( a ) Time-course analysis of recycled active α5β1 integrin in siCTL ECs versus siRAB11A ECs or siRAB11B ECs. RAB11B, but not RAB11A, silencing significantly impairs active α5β1 integrin recycling. Data are mean±s.e.m., n =3 independent experiments (two technical replicates for each experiment). ( b ) Confocal microscopy analysis of anti-active α5β1 integrin mAb SNAKA51 localization (red) in living confluent ECs (20 min of incubation). SNAKA51 + active α5β1 integrin localizes in fibrillar adhesion in siCTL and in siRAB11A, but not in siRAB11B ECs in which it accumulates in perinuclear punctae. The number of active α5β1 integrin-containing adhesions per 100 μm 2 of cell area was quantified in siCTL, siRAB11A and siRAB11B ECs. Data are mean values±s.e.m., n =20 cells per condition pooled from two independent experiments. ( c ) Confocal xz sectioning microscopy analysis of anti-active α5β1 integrin mAb SNAKA51 localization (red) in living confluent ECs (20 min of incubation). Quantitative analysis of apico-basal mean intensity ratio reveals that SNAKA51 + active α5β1 integrin localizes on the basolateral surface of siCTL and siRAB11A, but not of siRAB11B ECs, where it redistributes all around the cell surface. Data are mean values±s.e.m., n =20 cells per condition pooled from two independent experiments. ( d ) Confocal microscopy analysis of IST9 mAb + endogenous cellular ED-A FN (green) in confluent ECs. ED-A FN polymerizes into a fibrillar network in siCTL and siRAB11A, but not in siRAB11B ECs, where it accumulates in a perinuclear compartment. Relative amount of fibrillary ED-A FN area was calculated in siCTL, siRAB11A and siRAB11B ECs. Data are mean values±s.e.m., n =20 cells per condition pooled from 2 independent experiments. ( e ) Western blot analysis of soluble ED-A FN released by confluent ECs seeded on Transwell inserts. An equal percentage of apical and basolateral volumes of medium were collected after 72 h of culture from different wells of siCTL or siRAB11B ECs. Equal amounts of rabbit IgG were exogenously added to samples (spike normalization) for loading control purposes. Quantification of the ratio between apical or basolateral amount of ED-A FN released by siCTL over siRAB11B ECs. RAB11B silencing much more severely impairs basolateral than apical ED-A FN secretion. Data are mean±s.e.m., n =6 wells per condition pooled from three independent experiments. Scale bar, 50 μm ( d ), 20 μm ( b ), 5μm ( c ). ** P <0.01; *** P <0.001; Student's t -test.

Article Snippet: Rabbit polyclonal anti-RAB11A (TA324158) and RAB11B (TA346586) were from Origene, Rockville, MD, USA.

Techniques: Confocal Microscopy, Incubation, Microscopy, Western Blot, Control

Journal: eLife

Article Title: A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells

doi: 10.7554/eLife.56059

Figure Lengend Snippet: ( A ) Corin (green) and Rab11 proteins (red), including Rab11a, Rab11b, and dominant-negative (DN) mutants that were inhibitory to Rab11a and Rab11b, respectively, were co-expressed in MDCK cells. Immunostaining and confocal microscopy were used to analyze corin expression on apical and basolateral membranes, as indicated in X-Y and X-Z views. Each image represents the data from four experiments. Scale bars: 5 μm. ( B ) qRT-PCR analysis of Rab11a mRNA levels in MDCK cells transfected with two sets of shRNAs targeting the RAB11A gene (shRab11a1 and shRab11a2) or non-targeting control shRNAs (shNC). The data are mean ± SD from five experiments, analyzed by ANOVA. ( C ) Immunostaining of corin and CD320 in MDCK cells transfected with RAB11A -targeting shRNAs (shRab11a1 and shRab11b) or non-targeting shRNAs (shNC). X-Y and X-Z views are indicated. ( D ) A proposed model. Corin and CD320 (red dots) are transported from Golgi, likely via central recycling endosomes (CRE), to a Rab11a-positive sorting station, where the DSSDE motif is recognized for apical sorting via apical recycling endosomes (ARE) (left panel). Any pool of corin and CD320 that is endocytosed may also be re-sorted apically via Rab11a-positive endosomes. Mutations altering the DSSDE motif abolish such a mechanism, resulting in apical and basolateral expression of corin and CD320 mutants (right panel). Figure 8—source data 1. Source data for .

Article Snippet: Western blotting was used to verify Rab11a and Rab11b protein levels in the targeted cells with primary antibodies against Rab11a (Cell Signaling, 2413, 1:1000) and Rab11b (Boster, M04526, 0.5 μg/mL).

Techniques: Dominant Negative Mutation, Immunostaining, Confocal Microscopy, Expressing, Quantitative RT-PCR, Transfection, Control

Journal: eLife

Article Title: A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells

doi: 10.7554/eLife.56059

Figure Lengend Snippet: ( A ) Corin WT co-expressed with Rab11a, Rab11b, or dominant-negative (DN) Rab11a or Rab11b mutants in MDCK cells was analyzed by immunostaining and confocal microscopy. Quantitative data of the ratio of F BL /F Total from four experiments are shown. ( B ) Western blotting of Rab11a protein in MDCK cells transfected with non-targeting (shNC) and two sets of RAB11A -targeting (shRab11a1 and shRab11a2) shRNAs. GAPDH was used as a control. ( C ) Quantitative data of the ratio of F BL /F Total for corin and CD320 expression on apical and basolateral membranes in shRNA-transfected MDCK cells from four experiments. Statistical analysis was done with ANOVA. n.s., not significant vs . respective controls in each experiment. Figure 8—figure supplement 1—source data 1. Source data for . Figure 8—figure supplement 1—source data 2. Source data for .

Article Snippet: Western blotting was used to verify Rab11a and Rab11b protein levels in the targeted cells with primary antibodies against Rab11a (Cell Signaling, 2413, 1:1000) and Rab11b (Boster, M04526, 0.5 μg/mL).

Techniques: Dominant Negative Mutation, Immunostaining, Confocal Microscopy, Western Blot, Transfection, Control, Expressing, shRNA

Journal: eLife

Article Title: A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells

doi: 10.7554/eLife.56059

Figure Lengend Snippet: MDCK cells were transfected with non-targeting (shNC) or two sets of RAB11B -targeting (shRab11b1 and shRab11b2) shRNAs. Quantitative RT-PCR ( A ) and western blotting ( B ) were used to examine Rab11b mRNA and protein expression, respectively, in the shRNA-transfected MDCK cells. ( C ) Apical corin (top panels) and CD320 (lower panels) expression in the shRNA-transfected MDCK cells was examined by immunostaining and confocal microscopy with X-Y and X-Z views indicated. ( D ) Quantitative data of the ratio of F BL /F Total for corin and CD320 expression on apical and basolateral membranes in shRNA-transfected MDCK cells from four experiments. Statistical analysis was done with ANOVA. n.s., not significant vs . respective controls in each experiment. Key Resources Table. Information on genes, cell lines, plasmids and antibodies. Figure 8—figure supplement 2—source data 1. Source data for . Figure 8—figure supplement 2—source data 2. Source data for .

Article Snippet: Western blotting was used to verify Rab11a and Rab11b protein levels in the targeted cells with primary antibodies against Rab11a (Cell Signaling, 2413, 1:1000) and Rab11b (Boster, M04526, 0.5 μg/mL).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing, shRNA, Immunostaining, Confocal Microscopy

Journal: eLife

Article Title: A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells

doi: 10.7554/eLife.56059

Figure Lengend Snippet:

Article Snippet: Western blotting was used to verify Rab11a and Rab11b protein levels in the targeted cells with primary antibodies against Rab11a (Cell Signaling, 2413, 1:1000) and Rab11b (Boster, M04526, 0.5 μg/mL).

Techniques: Transfection, Construct, Plasmid Preparation