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:

Journal:

Article Title: Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton's Tyrosine Kinase

doi:

Figure Lengend Snippet: Ectopic expression of wild-type, but not K430E mutant, Btk leads to enhanced tyrosine phosphorylation of TFII-I. (A) TFII-I and either wild-type or K430E mutant Btk was coexpressed in COS cells, and TFII-I was pulled down by GST-agarose beads and probed with anti-P-Tyr (α-P-Tyr) antibody 4G10 in a Western blot analysis. The blot was stripped and reprobed with anti-TFII-I (α-TFII-I) antibody. The lysates were also tested for the expression of wild-type and K430E Btks. (B) For quantitation, these experiments were performed three times and the results are represented as graphs with error bars.

Article Snippet: The primary antibody, anti-TFII-I rabbit serum (IgG fraction, purified over Affigel Blue; Bio-Rad), or a nonspecific control (rabbit anti-human idiotype, generously provided by David Stollar, Tufts University School of Medicine) was diluted 1:10.

Techniques: Expressing, Mutagenesis, Western Blot, Quantitation Assay

Journal:

Article Title: Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton's Tyrosine Kinase

doi:

Figure Lengend Snippet: TFII-I and Btk associate in the cytoplasm. Wild-type Btk (WtBtk) or R28C or K430E mutant Btk was ectopically expressed in COS7 cells. Cytoplasmic extracts (normalized by total protein concentration) were prepared, the ectopically expressed Btk was immunoprecipitated by anti-HA antibody, and the coimmunoprecipitated endogenous TFII-I was visualized by anti-TFII-I (α-TFII-I) antibody. The blot was stripped and reprobed with anti-Btk (α-Btk) antibody.

Article Snippet: The primary antibody, anti-TFII-I rabbit serum (IgG fraction, purified over Affigel Blue; Bio-Rad), or a nonspecific control (rabbit anti-human idiotype, generously provided by David Stollar, Tufts University School of Medicine) was diluted 1:10.

Techniques: Mutagenesis, Protein Concentration, Immunoprecipitation

Journal:

Article Title: Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton's Tyrosine Kinase

doi:

Figure Lengend Snippet: Wild-type Btk, but not mutant Btks, potentiates TFII-I-dependent transcriptional stimulation of Vβ 5.2 in COS7 cells. (A) Transient transfection of COS7 cells. Shown are basal-level expression of the Vβ 5.2 promoter (−, lane 1) and expression in the presence of ectopic TFII-I alone (+TFII-I, lane 2), wild-type Btk (+Wt, lane 3), or xid mutant Btk (+R28C, lane 5). Cotransfection of wild-type Btk with TFII-I (TFII-I + Wt, lane 4), but not xid mutant Btk with TFII-I (+TFII-I + R28C, lane 6), further potentiates TFII-I-mediated activation of the Vβ 5.2 reporter. Western blotting of transfection extracts with an anti-Btk antibody (α-Btk) or an anti-TFII-I antibody (α-TFII-I) demonstrates equivalent levels of ectopic TFII-I expression in the indicated lanes. NS, nonspecific bands. (B) Wild-type Btk, but not kinase-deficient (K430E) Btk, potentiates TFII-I-mediated stimulation of the Vβ 5.2 promoter. The Vβ 5.2 promoter basal expression (−, lane 1) is stimulated by TFII-I (+TFII-I, lane 2). Neither wild-type (+Wt, lane 3) nor K430E mutant (+K430E, lane 5) Btk affects Vβ 5.2 promoter expression independently. Cotransfection of TFII-I with wild-type Btk (TFII-I + Wt, lane 4) but not kinase-deficient Btk (+TFII-I + K430E, lane 6) further potentiates TFII-I-mediated activation of the Vβ 5.2 promoter.

Article Snippet: The primary antibody, anti-TFII-I rabbit serum (IgG fraction, purified over Affigel Blue; Bio-Rad), or a nonspecific control (rabbit anti-human idiotype, generously provided by David Stollar, Tufts University School of Medicine) was diluted 1:10.

Techniques: Mutagenesis, Transfection, Expressing, Cotransfection, Activation Assay, Western Blot

Journal:

Article Title: Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton's Tyrosine Kinase

doi:

Figure Lengend Snippet: TFII-I interacts with both wild-type and K430E mutant Btks but not with R28C mutant Btk. (A) Normalization of extracts expressing ectopic TFII-I and Btk proteins. COS7 cells ectopically expressing either TFII-I alone (TFII-I) or together with HA-tagged wild-type Btk (TFII-I + Wt Btk), xid mutant Btk (TFII-I + R28C), or kinase-deficient Btk (TFII-I + K430E) were Western blotted with an anti-Btk antibody (Btk). The blot was then stripped and reprobed with an anti-TFII-I antibody (TFII-I). (B) Normalized extracts from panel A were employed for immunoprecipitation (I.P.) studies with an anti-HA (α-HA) antibody and probed either with an anti-TFII-I (α-TFII-I) antibody (left) or with an anti-Btk (α-Btk) antibody (right). The α-TFII-I blot (left) was stripped of immune complexes and reprobed with an anti-Btk antibody (Btk; right). Comparable amounts of Btk protein were precipitated with the anti-HA antibody from extracts ectopically expressing either wild-type or mutant Btk.

Article Snippet: The primary antibody, anti-TFII-I rabbit serum (IgG fraction, purified over Affigel Blue; Bio-Rad), or a nonspecific control (rabbit anti-human idiotype, generously provided by David Stollar, Tufts University School of Medicine) was diluted 1:10.

Techniques: Mutagenesis, Expressing, Western Blot, Immunoprecipitation

Journal:

Article Title: Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton's Tyrosine Kinase

doi:

Figure Lengend Snippet: TFII-I is constitutively associated with Btk in both human and murine B cells but dissociates from Btk upon anti-Ig antibody stimulation. (A) Whole-cell lysates prepared from human (Ramos) and murine (BAL-17) B cells were subjected to immunoprecipitation (IP) with an anti-Btk (α-Btk) antibody. A highly purified preparation of native TFII-I was used as a positive control. An anti-TFII-I (α-TFII-I) antibody recognizes two immunoreactive forms of endogenous TFII-I in each cell line, although in different ratios. Neither form is coprecipitated with a control antibody (Control), but both forms of endogenous TFII-I are coprecipitated by an anti-Btk antibody. (B) Cytoplasmic extracts were prepared from Ramos cells treated either with medium alone (−) or with anti-Ig (α-Ig) antibody, and endogenous TFII-I was coimmunoprecipitated with an anti-Btk antibody and visualized with an anti-TFII-I antibody. The blot was stripped and reprobed with anti-Btk antibody. (C) Cytoplasmic lysates from each treatment were analyzed by Western blot analysis for total cytoplasmic TFII-I and represent half of the amount used in panel B.

Article Snippet: The primary antibody, anti-TFII-I rabbit serum (IgG fraction, purified over Affigel Blue; Bio-Rad), or a nonspecific control (rabbit anti-human idiotype, generously provided by David Stollar, Tufts University School of Medicine) was diluted 1:10.

Techniques: Immunoprecipitation, Purification, Positive Control, Western Blot

Journal:

Article Title: Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton's Tyrosine Kinase

doi:

Figure Lengend Snippet: Localization of TFII-I in wild-type and xid mutant primary B cells. Localization of TFII-I in primary B cells by fluorescent staining and confocal microscopy. Wild-type (a) and xid mutant (b) splenic B cells were stained with an anti-TFII-I rabbit serum. Nuclear DNA was revealed by red propidium iodide staining (c and d). The two images were superimposed to generate panels e (wild type) and f (xid). In wild-type cells, TFII-I was predominantly cytoplasmic with some scattered nuclear staining (green in a and yellow in e). In xid B cells, the larger amount of nuclear green (b) and intense yellow (pseudocoloring showing concordance of red and green staining) in the superimposed image (f) indicated that these cells have more nuclear TFII-I. In three experiments, pixel counting revealed that the amount of nuclear TFII-I was 2.2-, 1.5-, and 2.3-fold greater in xid B cells than in wild-type B cells. The bottom panels are enlarged images of the central cells of panels e and f.

Article Snippet: The primary antibody, anti-TFII-I rabbit serum (IgG fraction, purified over Affigel Blue; Bio-Rad), or a nonspecific control (rabbit anti-human idiotype, generously provided by David Stollar, Tufts University School of Medicine) was diluted 1:10.

Techniques: Mutagenesis, Staining, Confocal Microscopy

Journal:

Article Title: Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton's Tyrosine Kinase

doi:

Figure Lengend Snippet: Nuclear distribution and tyrosine phosphorylation of TFII-I in wild-type (Wt) and xid mutant primary B cells. (A) Nuclear distribution of TFII-I in wild-type and xid mutant primary B cells. Shown is SDS-PAGE of whole-cell lysates (Whole Cell) derived from wild-type and xid mutant primary B cells, loaded according to equivalent cell numbers, and subsequent Western blotting with an anti-TFII-I (α-TFII-I) antibody (left) either in the absence or in the presence of anti-IgM (α-Ig) antibody stimulation. Also shown are nuclear extracts (Nuclear) from resting wild-type and xid mutant primary B cells (right) in the absence or presence of IgM (α-Ig) antibody activation. (B) Cytoplasmic and nuclear extracts from panel A were subjected to Western blot analysis with anti-P-Tyr (α-P-Tyr) antibody. The position of TFII-I was determined by running a purified preparation of native authentic TFII-I (data not shown).

Article Snippet: The primary antibody, anti-TFII-I rabbit serum (IgG fraction, purified over Affigel Blue; Bio-Rad), or a nonspecific control (rabbit anti-human idiotype, generously provided by David Stollar, Tufts University School of Medicine) was diluted 1:10.

Techniques: Mutagenesis, SDS Page, Derivative Assay, Western Blot, Activation Assay, Purification

Journal:

Article Title: Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton's Tyrosine Kinase

doi:

Figure Lengend Snippet: Model for Btk-dependent TFII-I function. A fraction of TFII-I is constitutively associated with Btk in the cytoplasm of wild-type resting B cells. Upon signaling through the Ig receptor, Btk is activated (Btk#), leading to tyrosine phosphorylation of TFII-I either directly or indirectly. While Btk# may localize to the plasma membrane to bind phospholipids, trigger calcium signaling, and activate diacyl glycerol (DAG), tyrosine-phosphorylated TFII-I is released from Btk# and translocates to the nucleus, where it is serine phosphorylated, perhaps through a Ras-dependent pathway. Although tyrosine phosphorylation of TFII-I may not be necessary for its nuclear import, it may be required for its maximal transcriptional activity. In xid B cells, TFII-I may not be constitutively associated with Btk and thus, increased amounts are in the nucleus.

Article Snippet: The primary antibody, anti-TFII-I rabbit serum (IgG fraction, purified over Affigel Blue; Bio-Rad), or a nonspecific control (rabbit anti-human idiotype, generously provided by David Stollar, Tufts University School of Medicine) was diluted 1:10.

Techniques: Activity Assay