Journal: The Journal of Cell Biology

Article Title: Interaction between FIP5 and SNX18 regulates epithelial lumen formation

doi: 10.1083/jcb.201011112

Figure Lengend Snippet: SNX18 is FIP5-binding protein. (A) FIP5 was immunopreciptated from HeLa cell lysates with anti-FIP5 antibody. The immunoprecipitate was then separated by SDS-PAGE and stained with Coomassie dye. Proteins listed in the figure were identified by at least two peptides from the anti-FIP5 immunoprecipitate, and were not present in the IgG control. (B) Human SNX18 sequence. Boxed regions indicate the peptides identified in proteomic analysis of the immunoprecipitate from A. (C) SNX18 or FIP5 were immunoprecipitated from MDCK cell lysates and immunoblotted with anti-SNX18, anti-FIP5, and anti-SNX9 antibodies. (D) HeLa cells were cotransfected with myc-SNX18 and FIP5-GFP. Cells were lysed, and myc-SNX18 was immunoprecipitated with anti-myc antibodies and blotted with anti-SNX18 and anti-GFP antibodies. (E) Schematic representation of the domains present in SNX9 and SNX18 proteins. Numbers between the SNX9 and SNX18 schematics indicate the percentage of homology between the corresponding domains of these proteins.

Article Snippet: Antibodies were affinity purified using recombinant SNX18 conjugated to Affigel (Bio-Rad Laboratories) and eluted with 0.1 M glycine buffer, pH 2.5.

Techniques: Binding Assay, SDS Page, Staining, Control, Sequencing, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Interaction between FIP5 and SNX18 regulates epithelial lumen formation

doi: 10.1083/jcb.201011112

Figure Lengend Snippet: FIP5 binds to SNX18-LC domain. (A and B) The affinity of FIP5 and SNX18 binding as determined by ITC. N.D., not detected. (C) Glutathione beads were coated with GST-SNX18, GST-SNX9, or GST alone and incubated with 6His-FIP5 or 6His-FIP3 in the presence or absence of a fivefold excess of Rab11a-GTP. The amount of bound 6His-FIP3 or 6His-FIP5 was determined by immunoblotting with anti-FIP3 or anti-FIP5 antibodies. (D) Glutathione beads were coated with GST-SNX18 or GST alone and incubated with 6His-FIP5 in the presence of increasing concentrations of soluble recombinant SNX9. The amount of bound 6His-FIP5 was determined by immunoblotting with anti-FIP5 antibodies. (E) Schematic representation of the FIP5-binding domain in SNX18 as determined by glutathione bead pull-down assays.

Article Snippet: Antibodies were affinity purified using recombinant SNX18 conjugated to Affigel (Bio-Rad Laboratories) and eluted with 0.1 M glycine buffer, pH 2.5.

Techniques: Binding Assay, Incubation, Western Blot, Recombinant

Journal: The Journal of Cell Biology

Article Title: Interaction between FIP5 and SNX18 regulates epithelial lumen formation

doi: 10.1083/jcb.201011112

Figure Lengend Snippet: FIP5 induces SNX18- and SNX9-dependent liposome tubulation. (A) SNX18 or FIP5 were incubated with PS/PC liposomes containing various phosphatidylinositides. Liposomes were then sedimented and levels of bound SNX18 or FIP5 were determined by Coomassie staining. (B) C- (FIP5 C-terminal) or N-terminal (FIP5-C2) domains of FIP5 were tested for their ability to bind PS/PC liposomes containing 5% PI(4,5)P 2 . The levels of bound proteins were determined by Coomassie staining. (C) SNX18 was incubated with PS/PC/PI(4,5)P 2 liposomes in the presence or absence of GST-FIP5. The levels of bound SNX18 and GST-FIP5 were determined by Coomassie staining. Black lines indicate the removal of intervening lanes for presentation purposes. (D) EM analysis of liposomes incubated with GST, GST-SNX9, GST-SNX18, 6His-FIP3, and 6His-FIP5. Arrows point to wide tubules (122.1 ± 32.9 nm, n = 10) and arrowheads point to narrow tubules (54.1 ± 18.1nm, n = 10). The asterisk marks short tubules induced by SNX18 alone.

Article Snippet: Antibodies were affinity purified using recombinant SNX18 conjugated to Affigel (Bio-Rad Laboratories) and eluted with 0.1 M glycine buffer, pH 2.5.

Techniques: Incubation, Liposomes, Staining

Journal: The Journal of Cell Biology

Article Title: Interaction between FIP5 and SNX18 regulates epithelial lumen formation

doi: 10.1083/jcb.201011112

Figure Lengend Snippet: SNX18 is required for the establishment of the apical lumen at the early stages of epithelial cyst formation. (A and B) MDCK-shSNX18 cells were grown for 9 d in the presence (A-c and A-d) or absence (A-a and A-b) of 1 µg/ml of dox. Cells were then fixed with 4% paraformaldehyde and stained with anti-gp135 (A-a and A-c) or anti-cingulin (A-b and A-d) antibodies. B shows the quantitation of epithelial cysts with a single lumen. Data shown are the means and standard deviations derived from three independent experiments (error bars). n is the number of cysts analyzed. Insets show dox+ cyst expressing myc-SNX18. (C–E) MDCK-shSNX18 cells were grown for 74 h in the presence (C-c, C-d, and C-f) or absence (C-a, C-b, and C-e) of 1 µg/ml of dox to pre-knockdown SNX18. Cells were then seeded in 3D cultures and grown for 24 h. Cells were fixed with 4% paraformaldehyde and stained with anti-gp135, anti-cingulin, or anti-SNX18 antibodies. E shows the quantitation of 3D cyst polarization at the two and four cell stages of the experiment shown in C. Data shown are the means and standard deviations derived from three independent experiments. n is the number of cysts analyzed. (D) The quantitation of fully matured (9 d) epithelial cysts with a single lumen in cells incubated with or without 1 µg/ml of dox added after 24 h in 3D cultures. Data shown are the means and standard deviations derived from three independent experiments (error bars). n is the number of cysts analyzed. Insets show the extent of SNX18 (green) knockdown in the presence of dox. Bars: (A) 8 µm; (B) 16 µm; (C) 3 µm; (D) 16 µm.

Article Snippet: Antibodies were affinity purified using recombinant SNX18 conjugated to Affigel (Bio-Rad Laboratories) and eluted with 0.1 M glycine buffer, pH 2.5.

Techniques: Staining, Quantitation Assay, Derivative Assay, Expressing, Knockdown, Incubation

Journal: The Journal of Cell Biology

Article Title: Interaction between FIP5 and SNX18 regulates epithelial lumen formation

doi: 10.1083/jcb.201011112

Figure Lengend Snippet: Proposed model of the roles of FIP5 and SNX18 in apical lumen formation and endosomal scission.

Article Snippet: Antibodies were affinity purified using recombinant SNX18 conjugated to Affigel (Bio-Rad Laboratories) and eluted with 0.1 M glycine buffer, pH 2.5.

Techniques: