Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Phosphorylation regulates FOXC2 function in vivo. (A) Endothelial cell-specific gain-of-function models for the analysis of FOXC2 phosphorylation. (B) Both models express comparable levels of the transgene, as evidenced by RT-PCR analysis of the indicated mRNAs from E15.5 lungs. Transgene expression was initiated at E13.5. (C) Macroscopic appearances of FOXC2ecGOF, pmFOXC2ecGOF, and control E15.5 embryos. (D and E) FOXC2 overexpression does not affect capillary sprouting. (F) Overexpression of FOXC2 but not pmFOXC2 promotes vascular remodeling in maturing capillaries. Note the increased capillary branching and density in FOXC2ecGOF embryos. Whole-mount staining of E15.5 head skin for pan-endothelial marker CD31 (green) and the transgene (red). The transgene expression was detected using anti-Myc antibody. Scale bars: 100 μm (D), 38 μm (E), 35 μm (F). (G) Quantification of vascular branching, density, and sprouting at the vascular front in the control, pmFOXC2ecGOF, and FOXC2ecGOF embryos. n = 3 per genotype. *, P < 0.05. n.s., nonsignificant.

Article Snippet: For immunofluorescence staining, we used mouse anti-Myc (clone 9E10; Santa Cruz Biotechnology), rabbit anti-Myc-Tag (clone 71D10; Cell Signaling Technology, used for transgene detection in vivo ), rat anti-mouse PECAM-1 (BD Pharmingen), rat anti-mouse FOXC2 ( 19 ), sheep anti-human FOXC2 (R&D Systems), and goat anti-human PROX1 (R&D Systems).

Techniques: In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing, Over Expression, Staining, Marker

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Analysis of FOXC2 phosphorylation. (A) Endogenous and recombinant human FOXC2 are similarly phosphorylated in primary LECs and immortalized cell lines. Cell lysates were treated (+) or not treated (−) with lambda protein phosphatase (λ-PPase) and analyzed by Western blotting with anti-FOXC2 or anti-Myc antibodies. (B) Schematic representation of FOXC2 phosphorylation sites. FHD, forkhead domain; TA, transactivation domains (5, 34); PD, phosphorylated domain. Phosphorylation sites identified by LC-MS/MS are shaded in red; phosphorylation sites identified by mutagenesis are shaded in yellow. Peptides detected by MS in tryptic and Glu-C digests are underlined in cyan and green, respectively. Amino acid numbering is the same as in the endogenous protein (NP_005242). (C) Substitution of eight phosphorylation sites in Myc-FOXC2 with alanine abolishes the phosphorylation-dependent electrophoretic mobility shift. Lysates of cells transfected with a plasmid expressing the phosphorylation-deficient mutant Myc-pmFOXC2 were treated (+) or not treated (−) with λ-PPase and analyzed by Western blotting with anti-Myc antibody. (D) FOXC2 phosphorylation-deficient mutant (pm) has increased electrophoretic mobility compared to the wild-type (wt) protein. Shown is Western blot analysis of lysates from HepG2 cells transduced with adenoviruses expressing Myc-FOXC2 or Myc-pmFOXC2.

Article Snippet: For immunofluorescence staining, we used mouse anti-Myc (clone 9E10; Santa Cruz Biotechnology), rabbit anti-Myc-Tag (clone 71D10; Cell Signaling Technology, used for transgene detection in vivo ), rat anti-mouse PECAM-1 (BD Pharmingen), rat anti-mouse FOXC2 ( 19 ), sheep anti-human FOXC2 (R&D Systems), and goat anti-human PROX1 (R&D Systems).

Techniques: Recombinant, Western Blot, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Electrophoretic Mobility Shift Assay, Transfection, Plasmid Preparation, Expressing, Transduction

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: FOXC2 interacts with peptidyl-prolyl cis/trans isomerase PIN1, alpha isoform of the regulatory subunit B of the protein phosphatase PP2A (PPP2R2A), and ERK1/2 protein kinases. (A) Coimmunoprecipitation assays with anti-Myc antibody demonstrate the association of Myc-FOXC2 with endogenous PIN1 in HeLa cells transduced with recombinant Ad-Myc-FOXC2. Shown is Western blot (WB) of anti-Myc immunoprecipitates consecutively probed with anti-Myc and anti-PIN1 antibodies. Control immunoprecipitation was performed from extracts of HeLa cells transduced with recombinant adenovirus expressing bacterial β-galactosidase (Ad-LacZ). (B) Myc-FOXC2 binds to endogenous PPP2R2A and ERK1/2 in HepG2 cells transduced with recombinant Ad-Myc-FOXC2. Coimmunoprecipitation assays were performed and analyzed as in A, except that anti-PPP2R2A and anti-total ERK1/2 antibodies were used for immunoblotting. (C) Immunocomplex kinase assays demonstrate that Myc-FOXC2 is phosphorylated in vitro by the coprecipitating endogenous ERK1/2 kinases. Shown is Western blot (WB) of anti-Myc immunoprecipitates incubated in the presence of [γ-32P]ATP and phosphorimage of the corresponding membrane. The blot was consecutively probed with anti-Myc, anti-total ERK1/2 and anti-active ERK1/2 (p-ERK1/2) antibodies. The identity of ERK1/2 was confirmed by immunocomplex kinase assays with anti-Myc antibody using lysates of HepG2 cells stimulated with PMA in the presence or absence of 10 mM U0126, a selective inhibitor of upstream MEK. (D) Inhibition of ERK1/2 does not modify the electrophoretic mobility of endogenous FOXC2 in LECs. Shown is a Western blot of total LEC lysates consecutively probed with anti-FOXC2 and anti-active ERK1/2 antibodies. (E) The electrophoretic mobility of endogenous FOXC2 changes after release from serum starvation-induced cell cycle arrest in LECs, suggesting CDK involvement in FOXC2 phosphorylation.

Article Snippet: For immunofluorescence staining, we used mouse anti-Myc (clone 9E10; Santa Cruz Biotechnology), rabbit anti-Myc-Tag (clone 71D10; Cell Signaling Technology, used for transgene detection in vivo ), rat anti-mouse PECAM-1 (BD Pharmingen), rat anti-mouse FOXC2 ( 19 ), sheep anti-human FOXC2 (R&D Systems), and goat anti-human PROX1 (R&D Systems).

Techniques: Transduction, Recombinant, Western Blot, Immunoprecipitation, Expressing, In Vitro, Incubation, Inhibition

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Phosphorylation regulates FOXC2-mediated transcription in primary LECs. (A) Immunofluorescent staining for Myc (green), lymphatic marker PROX1 (red), and DNA (blue) of LECs transduced with adenoviruses expressing wild-type Myc-FOXC2, phosphorylation-deficient mutant Myc-pmFOXC2, or control bacterial β-galactosidase (LacZ). Note that wild-type and mutant FOXC2 have similar expression levels and subcellular localization. Bars, 20 μm. (B) Phosphorylation regulates FOXC2 transcriptional activity. Gene expression profiling was performed on the adenovirus-transduced LECs shown in panel A. Genes whose expression changed >2-fold in response to the loss of FOXC2 phosphorylation (FDR < 0.01) are shown in orange (upregulated) and purple (downregulated) in the Volcano plot of significance against the fold change in gene expression. Vertical dotted lines mark the 2-fold change limits. (C) RT-PCR validation of the gene expression profiling results. Genes upregulated or downregulated >2-fold in response to the loss of FOXC2 phosphorylation are shown in orange and purple, respectively; genes affected <2-fold are shown in gray. No change in FOXC2 expression reflects equally efficient cell transduction with Ad-Myc-FOXC2 and Ad-Myc-pmFOXC2. The data are presented as log2-transformed fold change in gene expression normalized to a housekeeping gene (GAPDH). Horizontal dotted lines mark the 2-fold change limits. Shown are the means and standard deviations of triplicate determinations in a single experiment representative of two independent experiments. (D) Heat map representation of the differences in gene expression in response to the loss of FOXC2 phosphorylation. The left heat map shows expression levels of 57 of 59 genes downregulated >2-fold (FDR < 0.01) in Ad-Myc-pmFOXC2-transduced LECs compared to Ad-Myc-FOXC2-transduced LECs. The right heat map shows expression levels of 57 out of 88 genes upregulated >2-fold (FDR < 0.01) in the same cells. Three biological replicates are shown for each condition. The color key at the lower left corresponds to the mean-centered, arctan-transformed log2-fold change in gene expression falling within the range from −π/2 to π/2. Blue denotes genes with relative decreased expression; red denotes genes with relative increased expression.

Article Snippet: For immunofluorescence staining, we used mouse anti-Myc (clone 9E10; Santa Cruz Biotechnology), rabbit anti-Myc-Tag (clone 71D10; Cell Signaling Technology, used for transgene detection in vivo ), rat anti-mouse PECAM-1 (BD Pharmingen), rat anti-mouse FOXC2 ( 19 ), sheep anti-human FOXC2 (R&D Systems), and goat anti-human PROX1 (R&D Systems).

Techniques: Staining, Marker, Transduction, Expressing, Mutagenesis, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Transformation Assay

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Phosphorylation differentially regulates FOXC2 binding to genomic target sites in the context of native chromatin but not in vitro. We used genome-wide ChIP-chip to compare the binding of adenovirus-expressed wild-type Myc-FOXC2 and the phosphorylation-deficient mutant Myc-pmFOXC2 to physiological binding sites occupied by endogenous FOXC2 in primary LECs. Endogenous FOXC2 enrichment profiles are shown at the top of each panel. Purple peaks indicate FOXC2-enriched regions; their relative occupancies by Myc-FOXC2 and Myc-pmFOXC2 are shown in callout boxes in green and orange, respectively. Vertical axes represent MAT score. Binding sites are numbered as in Norrmén et al. (8); genomic coordinates refer to the hg18 human genome assembly. An unbound control region is shown in the lower right panel. The ChIP-chip results were validated by ChIP-qPCR with primers flanking ∼100-bp sequences within the FOXC2-enriched regions. The results are presented as the fold enrichment over the unbound control region. Green and orange bars correspond to wild-type Myc-FOXC2 and Myc-pmFOXC2, respectively. Shown are the means and standard deviations of triplicate determinations. An EMSA was used to compare the in vitro binding of adenovirus-expressed wild-type Myc-FOXC2, Myc-pmFOXC2, and deletion mutant Myc-FOXC2 D219-366 to naked dsDNA from the ChIP-enriched regions or the unbound control region. Binding specificity was controlled with adenovirus-expressed bacterial β-galactosidase (LacZ) and anti-Myc antibody. Asterisks indicate the positions of the antibody-supershifted complexes.

Article Snippet: For immunofluorescence staining, we used mouse anti-Myc (clone 9E10; Santa Cruz Biotechnology), rabbit anti-Myc-Tag (clone 71D10; Cell Signaling Technology, used for transgene detection in vivo ), rat anti-mouse PECAM-1 (BD Pharmingen), rat anti-mouse FOXC2 ( 19 ), sheep anti-human FOXC2 (R&D Systems), and goat anti-human PROX1 (R&D Systems).

Techniques: Binding Assay, In Vitro, Genome Wide, ChIP-chip, Mutagenesis

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) (A) and immunoblotting (B) analysis of VANGL2 mRNA and protein level change in A549 cells infected with vesicular stomatitis virus (VSV) [multiplicity of infection (MOI) of 0.5] for 0 to 16 hours. ( C ) Immunoblotting analysis of VANGL2 protein level changes in wild-type (WT) and Ifnar −/− peritoneal macrophages (PEMs) infected with VSV (MOI of 0.5) for the indicated times. ( D and E ) Luciferase reporter assays analyzing IFN-β or IFN-stimulated response element (ISRE) promoter activity of human embryonic kidney (HEK) 293T cells transfected with increasing amounts (wedge represents 300 and 500 ng) of HA-VANGL2 or empty vector (EV) for 24 hours, followed by treatment with or without VSV (MOI of 0.5) (D) or poly(I:C) (E) for 12 hours, respectively. ( F to J ) Immunoblotting analysis (F and I) of total and phosphorylated IRF3 and RT-PCR analysis (G), (H), and (J) of indicated gene expression in HEK293T (F) to (H) or A549 (I) and (J) cells transfected with FLAG-VANGL2 or EV for 24 hours, followed by VSV (MOI of 0.5) infection at indicated time points. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( K and L ) Fluorescence microscopy analysis (K) and flow cytometric analysis (L) of the replication of VSV–enhanced green fluorescent protein (eGFP) in HEK293T cells transfected with EV or increasing HA-VANGL2 at indicated dose for 24 hours, followed by treatment with or without VSV-eGFP (MOI of 0.5) infection at indicated time points. Numbers adjacent to the outlined areas indicate percentages of GFP + cells. NC, negative control. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test. ns, not significant.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Infection, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Expressing, Fluorescence, Microscopy, Negative Control

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A ) Luciferase activity in HEK293T cells transfected with scrambled ( Scr ) small interfering RNA (siRNA) or siRNA-targeting VANGL2 for 24 hours and then transfected with an IFN-β luciferase (IFN-β luc) for 24 hours, followed by treatment with or without VSV (MOI of 0.5), poly(I:C), or poly (dA:dT) for 12 hours. ( B and C ) RT-PCR (B) and immunoblotting (C) analysis of VSV (MOI of 0.5)–infected THP-1 cells transfected with Scr siRNA or VANGL2 -specific siRNA at indicated time points. ( D ) Heatmap view of top and bottom gene list of RNA-sequence data sets. Microarray analysis for total RNA was performed for Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + bone marrow–derived macrophages (BMDMs) with or without VSV infection. ( E ) VANGL2 regulates antiviral response-relevant target genes, presented as a volcano plot of genes with differential expression after VSV infection in Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs. FC, fold change. ( F ) Gene ontology (GO) enrichment analysis of the VANGL2-dependent genes in (E) (−log 2 P values). ( G ) Heatmap showing the change of indicated ISGs in Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs with or without VSV infection. ( H to L ) RT-PCR analysis of Ifnb (H), Isg56 (I), and VSV-G (K) mRNA expression, IFN-β enzyme-linked immunosorbent assay (ELISA) (J), and immunoblotting (L) analysis of total and phosphorylated IRF3 using Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Luciferase, Activity Assay, Transfection, Small Interfering RNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Infection, Sequencing, Microarray, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Luciferase reporter assays analyzing IFN-β (A) or ISRE (B) promoter activity of HEK293T cells transfected with the Flag-tagged indicated plasmids along with EV or increasing amounts (from 100 to 200 ng) of HA-VANGL2. ( C ) Co-immunoprecipitation (co-IP; with anti-FLAG) and immunoblotting analysis using protein lysates of HEK293T cells transfected with indicated plasmids. WCL, whole cell lysates. ( D ) Co-IP (with anti-TBK1) and immunoblotting analysis using endogenous proteins lysates of control and VSV (MOI of 0.5, 12 hours)–infected BMDMs. ( E ) Control and VSV (MOI of 0.5, 12 hours)–infected BMDMs were labeled with the indicated antibodies and analyzed via confocal microscopy. Red, VANGL2 signal; green, TBK1 signal; blue, 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 20 μm. ( F ) Quantitative analysis of the colocalization in (E). ( G ) Co-IP (with anti-VANGL2) and immunoblotting analysis using unsorted, cytosolic, and membrane lysates of THP-1 cells with or without VSV infection for 12 hours. ( H ) Schematic mapping of VANGL2. ( I ) Co-IP and immunoblotting analysis using lysates from HEK293T cells transfected with MYC-VANGL2 and its truncations along with FLAG-TBK1. ( J ) Co-IP (with anti-FLAG) and immunoblotting analysis using lysates from HEK293T cells transfected with vectors for HA-PKBD along with FLAG-TBK1. PkBD, the Prickle-binding domain. ( K and L ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with MYC-VANGL2 and its deletions along with FLAG-TBK1 (K) or infected with VSV (L) (MOI of 0.5). ( M and N ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with increasing amounts (from 300 to 500 ng) of HA-PKBD or EV along with FLAG-TBK1 (M) or infected with VSV (N) (MOI of 0.5). Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Luciferase, Activity Assay, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Infection, Labeling, Confocal Microscopy, Binding Assay

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) RT-qPCR (A) and immunoblotting (B) analysis of TBK1 mRNA and protein level change in HEK293T cells transfected with HA-TBK1, and increasing amounts of MYC-VANGL2. ( C ) Immunoblotting analysis of total and phosphorylated TBK1 using Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. ( D and E ) Immunoblotting analysis of total and phosphorylated TBK1 using THP-1 (D) or PBMCs (E) transfected with Scr siRNA or VANGL2 siRNA for 24 hours and then infected with VSV (MOI of 0.5) for indicated time. ( F ) Immunoblotting analysis of protein extracts of HEK293T cells treated with CHX (100 μg/ml) for 12 hours, followed by treatment with MG132 (10 μM), Baf A1 (0.2 μM), or both for 6 hours. ( G ) Immunoblotting analysis of HEK293T cells transfected with indicated plasmids for 24 hours, followed by treatment with MG132 (10 μM), 3-MA (10 mM), or Baf A1 (0.2 μM) for 6 hours. ( H ) Immunoblotting analysis using lysates from HEK293T cells transfected with FLAG-VANGL2 or FLAG-EV for 24 hours, followed by treatment with rapamycin (250 nM) for indicated time. ( I ) Immunoblotting analysis using lysates from Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs treated with EBSS for indicated time. ( J ) Immunoblotting analysis of WT, BECLIN -KO, or ATG5 -KO HEK293T cells transfected with indicated plasmids. ( K ) Control and VSV (MOI of 0.5, 12 hours)–infected BMDMs were labeled with the indicated specific antibodies and analyzed via confocal microscopy. Red, VANGL2 signal; green, TBK1 signal; violet, LAMP1 signal; blue, DAPI. Scale bars, 20 μm. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Infection, Labeling, Confocal Microscopy

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Co-IP (with anti-FLAG) and immunoblotting analysis using lysates from HEK293T cells transfected with indicated Flag-tagged cargo receptors along with HA-TBK1 (A) or HA-VANGL2 (B). ( C ) Cell lysates were harvested after Baf A1 (0.2 μM) treatment (6 hours) for co-IP (with anti-FLAG) and immunoblotting analysis of HEK293T cells transfected with HA-TBK1, FLAG-OPTN, and MYC-VANGL2. ( D ) Immunoblotting analysis of WT and OPTN −/− HEK293T cells transfected with FLAG-TBK1 and MYC-EV or MYC-VANGL2 for 24 hours. ( E ) Luciferase reporter assays analyzing IFN-β promoter activity of WT, OPTN -KO, NDP52- KO, p62 -KO, or TOLLIP -KO HEK293T cells transfected with FLAG-TBK1, together with increasing amounts (wedge represents 300 and 500 ng) of HA-VANGL2 for 24 hours. ( F and G ) A549 (F) or BMDMs (G) cells were infected with VSV (MOI of 0.5), and protein lysates were harvested for IP using an anti-OPTN antibody. ( H to J ) Immunoblotting (H) and RT-PCR (I) and (J) analysis of WT and OPTN -KO HEK293T cells transfected with FLAG-EV or FLAG-VANGL2 for 24 hours, followed by treatment with or without VSV (MOI of 0.5) infection for 12 hours. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. *** P < 0.001 and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection, Luciferase, Activity Assay, Infection, Reverse Transcription Polymerase Chain Reaction

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with FLAG-TBK1 and HA-tagged WT ubiquitin (HA-Ub) or its mutants, together with MYC-EV or MYC-VANGL2 for 24 hours, followed by treatment with Baf A1 (0.2 μM) for 6 hours, followed by co-immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA antibody. ( B ) IP (with anti-TBK1) and immunoblotting analysis using indicated antibodies of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + BMDMs infected with VSV (MOI of 0.5) for the indicated times. ( C ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with Scr shRNA or other E3 ligase–specific shRNAs for 24 hours, followed by transfected with FLAG-TBK1, together with EV or HA-VANGL2 for 24 hours. ( D ) HEK293T cells were transfected with Scr shRNA or TRIP- specific shRNA for 24 hours, followed by transfected with FLAG-EV or FLAG-VANGL2 for 24 hours; protein lysates were harvested after VSV (MOI of 0.5) infection for 12 hours and Baf A1 (0.2 μM) treatment for 6 hours for IP (with anti-TBK1) and immunoblotting analysis using indicated antibodies. ( E ) Luciferase reporter assays analyzing IFN-β promoter activity of HEK293T cells transfected with WT FLAG-TBK1 or its K323R, K341R, K344R, and K372R mutant, together with MYC-EV or MYC-VANGL2 for 24 hours. ( F ) Co-IP (with anti-FLAG) and immunoblotting analysis of HEK293T cells transfected with WT FLAG-TBK1 or its K323R, K341R, K344R, and K372R mutant, together with HA-K48–linked ubiquitin and MYC-EV or MYC-VANGL2; cell lysates were harvested after Baf A1 (0.2 μM) treatment for 6 hours. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Luciferase, Activity Assay, shRNA, Mutagenesis, Co-Immunoprecipitation Assay

Journal: Science Advances

Article Title: VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation

doi: 10.1126/sciadv.adg2339

Figure Lengend Snippet: ( A and B ) Weight (A) and survival (B) of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + mice ( n = 6 mice per group) after intraperitoneal injection of VSV [1 × 10 8 plaque-forming units (PFU) per mouse]. ( C ) ELISA for IFN-β in serum of Vangl2 fl/fl Lyz2 -Cre − and Vangl2 fl/fl Lyz2 -Cre + mice treated with phosphate-buffered saline (PBS) or infected with VSV (1 × 10 8 PFU per mouse) via intraperitoneal injection for 18 hours. ( D and E ) RT-PCR analysis of Ifnb (D) or VSV-G (E) mRNA in the spleen (left), lungs (center), and liver (right) from mice, as in (C). ( F ) Representative hematoxylin and eosin–stained images of lung sections from mice as in (C). Scale bars, 50 μm. ( G ) Graphical abstract to illustrate how VANGL2 negatively regulates IFN-I signaling upon virus infection. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 as determined by unpaired Student’s t test.

Article Snippet: Primary antibodies used for IP and immunoblot (IB) analysis are as follows: anti-TRAF3IP3 (no. ab243711, Abcam), anti-VANGL2 (C-2) (no. sc-515187, Santa Cruz Biotechnology), anti–phosphor-TBK1/NAK (Ser 172 ) (no. 5483, Cell Signaling Technology), anti-TBK1/NAK (no. 3013, Cell Signaling Technology), anti–phosphor-IRF3 (Ser 396 ) (no. 4947, Cell Signaling Technology), anti-IRF3 (no. 11904, Cell Signaling Technology), anti-FLAG (M2) (no. A8592, Sigma-Aldrich), anti-hemagglutinin (HA) (C29F4) (no. 5017, Cell Signaling Technology), anti-MYC (9B11) (no. 2276, Cell Signaling Technology), anti-p62 (D5L7G) (no. 88588, Cell Signaling Technology), anti-OPTN (EPR20654) (no. ab213556), anti-NDP52 (no. 12229-1-AP, Proteintech), anti-TOLLIP (no. 11315-1-AP, Proteintech), anti-ATG5 (no. 10181-2-AP, Proteintech), anti-BECLIN1 (no. 11306-1-AP, Proteintech), anti–Na,K-depenent adenosine triphosphatase (Na,K-ATPase) (D4Y7E) (no. 23565, Cell Signaling Technology), anti-Ub (P4D1) (no. 3936, Cell Signaling Technology), anti-K48-Ub (D9D5) (no. 8081, Cell Signaling Technology), anti-TUBULIN (210-444h) (no. sc-5274, Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (G-9) (no. sc-365062, Santa Cruz Biotechnology).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Infection, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Distribution of Nedd4-2 and TRPV6 in rat intestinal tract. A, RT-PCR results showing expression of TRPV6, Nedd4, and Nedd4-2 in rat intestine at the mRNA level. All PCR experiments were performed for 25 cycles. B, anti-Nedd4-2 antiserum specifically recognized Nedd4-2 but not Nedd4 exogenously expressed in X. laevis oocytes by Western blot analysis. A band with the same size as Nedd4-2 and two bands of lower molecular weight were recognized by this antiserum in rat colon lysate. C, immunofluorescent staining of oocytes injected with cRNAs for TRPV6, Nedd4-2, Nedd4, or water (as controls) with antisera against TRPV6 and Nedd4-2, respectively. TRPV6 and Nedd4-2 antisera recognized TRPV6 and Nedd4-2 expressed in the plasma membrane and cytoplasma of oocytes without specific staining in water or Nedd4 cRNA-injected oocytes. Scale bar, 50 μm. D, immunofluorescent staining of rat intestine tissues with TRPV6 and Nedd4-2 antisera in adjacent sections. TRPV6 antiserum labeled strongly in apical membrane in all segments of rat intestine tested. Nedd4-2 antiserum exhibited weak intracellular staining in duodenum and jejunum and strong staining in ileum, cecum, and colon. Preimmune serum (for control of TRPV6 staining) and secondary antibody alone (for control of Nedd4-2 staining) exhibited no specific staining in colon, respectively. Magnification was ×250 and ×800 for regular and enlarged sections, respectively.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Molecular Weight, Staining, Injection, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Nedd4-2 and Nedd4 decreased Ca2+ uptake, Na+ current, and protein abundance of TRPV6 and TRPV5 in X. laevis oocytes. A, Ca2+ uptake values of control oocytes or oocytes expressing Nedd4-2, Nedd4, TRPV6, or TRPV6 together with Nedd4-2 or Nedd4. Data are presented as means ± S.E. (error bars) of six independent experiments. *, p < 0.01 versus TRPV6 alone group. Representative Western blot analyses for proteins extracted from different groups using antisera against TRPV6 and β-actin (as loading control) are shown (bottom). B, current-voltage (I-V) plots of Na+-evoked currents in oocytes expressing TRPV6 alone or together with Nedd4-2 or Nedd4. Oocytes expressing Nedd4-2 or Nedd4 exhibited negligible currents similar to water-injected oocytes (not shown). Data are expressed as means ± S.E. of 14 oocytes in each group from four independent experiments. C, Ca2+ uptake in control oocytes (water-injected) or oocytes injected with TRPV5 cRNA alone or together with Nedd4-2 and Nedd4, respectively. Data are presented as means ± S.E. of six independent experiments. *, p < 0.01 versus TRPV5 alone group. TRPV5 protein level was decreased by Nedd4-2 or Nedd4 (bottom). D, current-voltage plots of Na+-evoked currents in oocytes expressing TRPV5 alone or together with Nedd4-2 or Nedd4. Data are expressed as means ± S.E. of 18 oocytes in each group from three independent experiments. All experiments were performed at 2 days after injection of cRNAs.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Expressing, Western Blot, Injection

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Nedd4-2 dose-dependently decreased TRPV6 protein abundance (A) and TRPV6-mediated Ca2+ uptake (B). Groups of X. laevis oocytes were injected with 12.5 ng of TRPV6 cRNA with 0, 3.1, 6.3, 12.5, or 25 ng of Nedd4-2 cRNA, and Ca2+ uptake experiments were performed 2 days later. Data are presented as a percentage of Ca2+ uptake of the group injected with TRPV6 cRNA alone. Representative Western blot analyses show that TRPV6 protein level decreased as Nedd4-2 protein level increased, and β-actin (loading control) was at a similar level in all of the groups (A). Error bars, S.E.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Nedd4-2 decreased TRPV6 stability. X. laevis oocytes were injected with 12.5 ng of TRPV6 cRNA alone or together with 12.5 ng of Nedd4-2 cRNA. Two days after injection, the stability of TRPV6 protein was examined by treating the oocytes with 100 μg/ml cycloheximide, an inhibitor of protein synthesis, for 0, 3, 6, or 9 h. A, representative Western blot analyses show the level of TRPV6 proteins at each time point in the presence and absence of Nedd4-2. The level of β-actin determined by Western blotting was used as a control for equal loading. B, assessment of the linear range of TRPV6 band intensity. Oocyte lysates containing TRPV6 protein were loaded at varying levels in different lanes and were run together with samples in A, and the linear range of TRPV6 protein level and band intensity were determined. Shown are a representative Western blot for TRPV6 (top) and the derived relationship of band intensity and TRPV6 protein level (bottom). TRPV6 band intensity is expressed as a percentage of that at 100% input. C, rate of TRPV6 protein degradation in the presence or absence of Nedd4-2. Protein level is expressed as a percentage of band intensity at time 0. Data from seven blots are shown as means ± S.E. (error bars). *, p < 0.05 versus the TRPV6 alone group.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Injection, Western Blot, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: The ubiquitin E3 ligase activity of Nedd4-2 was essential to its inhibitory effect on TRPV6. A, ubiquitination of TRPV6 was enhanced by Nedd4-2. Oocytes were injected with cRNA for TRPV6, Nedd4-2, and HA-tagged ubiquitin (HA-Ub) alone or in combination. Twenty-four hours after injection, oocytes were treated with 25 μm MG132 for 5 h before harvesting. TRPV6 was immunoprecipitated (IP) with a TRPV6 antiserum and immunoblotted (IB) with anti-HA to detect ubiquitinated TRPV6. A Western blot with TRPV6 antiserum is shown at the bottom. B, ligase activity of Nedd4-2 was necessary for the down-regulation of TRPV6. TRPV6 was expressed alone or together with WT Nedd4-2 or Nedd4-2 C922S mutant in oocytes. Data are presented as means ± S.E. (error bars) of three independent experiments. *, p < 0.01 versus TRPV6 alone.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Activity Assay, Injection, Immunoprecipitation, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Nedd4-2 did not increase the endocytosis TRPV6 but facilitated the proteasomal degradation of TRPV6. A, surface TRPV6 abundance was decreased by Nedd4-2. Oocytes expressing TRPV6 alone or with Nedd4-2 and control oocytes were probed with an antiserum that recognizes the first extracellular loop of TRPV6. The oocytes were then fixed for 1 h at 4 °C and then incubated with an HRP-coupled secondary antibody. Chemiluminescence signals of individual oocytes developed with HRP substrate were detected using a luminometer. The values are expressed as percentages of the chemiluminescence signal level of the TRPV6 alone group. Data from 40–57 oocytes in each group from two batches of oocytes are presented as mean ± S.E. (error bars). *, p < 0.01 versus TRPV6 alone group. B, Nedd4-2 did not alter the endocytosis of surface TRPV6. Oocytes were treated with the same maneuver as in A except that they were incubated for 0, 15, and 30 min for endocytosis at room temperature before incubation with secondary antibody. The values are expressed as percentages of the chemiluminescence signal levels at time 0 of the TRPV6 group or the TRPV6 plus Nedd4-2 group. Background values of water-injected control groups were subtracted. Data from 18–61 oocytes in each group from two batches of oocytes are presented as mean ± S.E. C, effects of proteasome inhibitor MG132 and lysosome inhibitor chloroquine on the regulation of TRPV6 by Nedd4-2. Oocytes expressing TRPV6 alone or with Nedd4-2 were incubated with 25 μm MG132 or 100 μm chloroquine for 5 h before Ca2+ uptake and Western blot experiments were performed. Data are presented as means ± S.E. of three independent experiments. *, p < 0.01 versus the TRPV6 plus Nedd4-2 group; NS, not statistically significant (p > 0.01).

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Expressing, Incubation, Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Assessment of Nedd4-2 domains on TRPV6. A and B, WT Nedd4-2 and Nedd4-2 constructs lacking C2 (ΔC2, without amino acids 1–166), WW1-2 (ΔWW1-2, without amino acids 194–398), WW3-4 (ΔWW3-4, without amino acids 478–561), or HECT (ΔHECT, without amino acids 618–955) were co-injected with TRPV6, and the Ca2+ uptake and TRPV6 protein level were determined 2 days later. A, all cRNAs were injected at 12.5 ng/oocyte; B, WT and ΔWW1-2 were injected at 1.6 ng/oocyte because ΔWW1-2 construct expression made oocytes unhealthy. *, p < 0.01 versus WT plus TRPV6 group. β-Actin was detected by Western blot analysis as a loading control. Only the ΔHECT construct was unable to inhibit TRPV6. C, HECT domain was necessary for the association between Nedd4-2 and TRPV6. Top, immunoblotting (IB) with the anti-HA antibody showed the expression of HA tagged wild-type and ΔHECT Nedd4-2 constructs. Bottom, HA-tagged Nedd4-2 proteins co-immunoprecipitated (IP) with TRPV6 proteins were detected by anti-HA antibody. HECT domain deletion abolished the association between Nedd4-2 and TRPV6. Error bars, S.E.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Construct, Injection, Expressing, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: TRPV6 directly interacted with Nedd4-2. A, autoradiography of GST fusion proteins with the Nedd4-2 C2 region (amino acids 1–166, including the C2 domain (amino acids 21–124)), WW1-2 region (amino acids 167–438), WW3-4 region (amino acids 439–590), or HECT region (amino acids 591–955, including the HECT domain 618–955) probed with 35S-labeled TRPV6 N-terminal (top) or C-terminal region (middle). Purified GST fusion proteins stained with Coomassie Blue are shown at the bottom. Note that the bands with lower molecular weights were present in lanes loaded with WW1-2 and HECT fusion proteins (top bands), respectively. The nature of these bands was unknown, but they may represent degraded products. B, autoradiography of individual GST fusion Nedd4-2 WW domains probed with 35S-labeled TRPV6 N-terminal (top) or C-terminal (middle) region. Purified GST fusion WW domains stained with Coomassie Blue are shown at the bottom. C, autoradiography of in vitro synthesized 35S-labeled TRPV6 N-terminal region (1–327 amino acids) and C-terminal region (578–725 amino acids) that were used as probes for A and B. D, autoradiography of GST fusion C-terminal region of TRPV6 (amino acids 597–725; the fusion protein was close to the 43 kDa marker) (top) probed with 35S-labeled Nedd4-2 full-length, ΔC2, ΔWW1-2, ΔWW3-4, and ΔHECT constructs (bottom) described in the legend to Fig. 7. The amounts of different probes were adjusted to the same level for each experiment in A, B, and D based on the band intensity.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Autoradiography, Labeling, Purification, Staining, In Vitro, Synthesized, Marker, Construct

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Asp204 and Asp376 of Nedd4-2 were critical to the interaction between TRPV6 N-terminal region and WW1 and WW2 domains, respectively. A, sequence alignment of four WW domains of Nedd4-2. *, amino acid residues in WW1 domain that are conserved in WW2 but not in WW3 and WW4 domains. B–D, Far Western analyses of key amino acid residues of WW1 in its interaction with TRPV6 terminal regions. GST fusion WW1, WW2, and WW4 peptides, including the WT and constructs with the indicated mutations, were probed with the 35S-labeled TRPV6 N-terminal region (amino acids 1–327; top) and C-terminal region (amino acids 578–725; middle), respectively. Purified GST fusion WW peptides stained with Coomassie Blue are shown at the bottom. B, D204H mutation in WW1 significantly attenuated the interaction between the TRPV6 N-terminal and WW1 domain. C, D204A mutation in WW1 also attenuated the interaction between the TRPV6 N-terminal region and the WW1 domain. The H539D mutation in the WW4 domain increased the interaction between the TRPV6 N-terminal region and WW4 domain. His539 in WW4 is the counterpart of Asp204 in WW1. D, D376H mutation in the WW2 domain also attenuated the interaction between the TRPV6 N-terminal region and WW2 domain. Asp376 in WW2 is the counterpart of Asp204 in WW1.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Sequencing, Western Blot, Construct, Labeling, Purification, Staining, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: D204H mutation in Nedd4-2 enhanced its ability to inhibit TRPV6. Top, TRPV6-mediated Ca2+ uptake in oocytes expressing TRPV6 alone or together with WT Nedd4-2, D204H, D376H, or D204H/D376H mutant. Oocytes injected with water (Water) were used as control. Data are presented as means ± S.E. (error bars) of three independent experiments. *, p < 0.01 versus TRPV6 alone group; #, p < 0.01 versus TRPV6 plus wild-type Nedd4-2 group. Representative Western blot analyses using antibodies against TRPV6, Nedd4-2, and β-actin are shown at the bottom.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Mutagenesis, Expressing, Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

doi: 10.1074/jbc.M110.175968

Figure Lengend Snippet: Nedd4-2 with D204H/D376H double mutations increased TRPV6 ubiquitination without compromising its association with TRPV6. A, association of TRPV6 with HA-Nedd4-2 and HA-Nedd4-2 D204H/D376H in oocytes. Top, immunoblotting (IB) with the anti-HA antibody indicates the equal level of wild-type HA-Nedd4-2 and HA-Nedd4-2 containing the D204H/D376H mutation. HA-Nedd4-2 proteins (wild type or mutant) co-immunoprecipitated with TRPV6 proteins were detected by anti-HA antibody. Bottom, intensity of the co-immunoprecipitated Nedd4-2 band was significantly decreased by the D204H/D376H mutation. Data from four experiments were normalized against band intensity of wild-type or mutant Nedd4-2. *, p < 0.01. AU, arbitrary unit. B, Nedd4-2 D204H/D376H mutant increased the ubiquitination of TRPV6 compared with the wild-type Nedd4-2. Details of the experiments were similar to those described in the legend to Fig. 5A. Error bars, S.E.

Article Snippet: Purification of GST Fusion Protein Nedd4-2 C2, WW1, WW2, WW3, WW4, and HECT domains were amplified by PCR and then subcloned into pGEX-6P-1 vector (Amersham Biosciences) and transferred into competent Escherichia coli BL21 by the heat shock method.

Techniques: Western Blot, Mutagenesis, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways

doi: 10.1074/jbc.RA120.012635

Figure Lengend Snippet: Malaysia/B HA is not activated by murine tPA, uPA, LTF, NSP4, CFB, tryptase ϵ, and HGFA. A, examination of HA cleavage by soluble proteases present in cell supernatants. HEK293 cells with transient expression of Malaysia/B HA were incubated with cleared protease containing HEK293 cell supernatants as described under “Experimental procedures” (left) or recombinant rKLK8 (right). Treatment of HA-expressing cells with buffer (w/o) or trypsin was used as control. Cell lysates were analyzed for HA cleavage by immunoblotting. β-Actin was used as loading control. B, MDCK cells with transient protease expression were infected with Malaysia/B at a low MOI of 0.01 and incubated for 24 h to allow multicycle viral replication. Cells transfected with empty vector (ev) or murine TMPRSS2-expressing plasmid were used as control. Virus spread was visualized by immunostaining of infected cells against NP. C, expression analysis of tryptase ϵ_DDDDK mutant in HEK293 cells with or without enterokinase treatment. Supernatants of cells transfected with empty vector (ev) or tryptase ϵ_DDDDK-encoding plasmid were concentrated (5×) at 48 h post-transfection and analyzed by SDS-PAGE and Western blotting using tryptase ϵ–specific antibodies. Zymogen and mature form are indicated by filled and open arrowheads, respectively. D, examination of HA cleavage by tryptase ϵ. HEK293 cells expressing Malaysia/B HA were incubated with tryptase ϵ_DDDDK mutant–containing cell supernatants treated with or without enterokinase (10 IU). Treatment of HA-expressing cells with trypsin was used as control. Cell lysates were analyzed for HA cleavage. E, expression analysis of HGFA in HEK293 supernatants with and without matriptase treatment. At 48 h post-transfection with empty vector (ev) or HGFA-encoding plasmid cell supernatants were concentrated (5×), treated with or without matriptase (5.0 μg/ml) for 1 h at 37 °C, and analyzed by immunoblotting using a FLAG-specific antibody. Zymogen and mature form are indicated by filled and open arrowheads, respectively. F, examination of HA cleavage by HGFA. HEK293 cells co-transfected with plasmids encoding Malaysia/B HA and either empty vector (w/o) or HGFA-encoding plasmid were incubated with exogenous matriptase or trypsin (0.5 μg/ml each) or remained untreated for 24 h. Cell lysates were analyzed for HA cleavage by Western blotting.

Article Snippet: The cDNA of the HA gene of B/Malaysia/2506/2004 was cloned from viral RNA by RT-PCR using HA-specific primers and subsequently subcloned into pCAGGS expression plasmid using EcoRI and NotI restriction sites. pCMV6-Entry expression plasmids encoding murine proteases with a C-terminal Myc-DDK tag were obtained from OriGene Technologies: CFB (MR210521), HGFA (MR216475), hepsin (MR219750), LTF (MR210170), tPA (MR208868), uPA (MR225747), tryptase ϵ (MR204321), NSP4 (MR217041), prostasin (MR223227), matriptase (MR222240), TMPRSS4 (MR206946), and TMPRSS13 (MR220731). pCAGGS plasmids encoding human or murine TMPRSS2 with C-terminal FLAG epitope have been described previously ( 6 , 15 ). pCAGGS encoding human prostasin was described previously ( 31 ). pcDNA6.2/C-emGFP-HPN encoding human hepsin (Human ORFeome Collaboration, Clone ID: 100004749) was generously provided by the Juha Klefström laboratory (University of Helsinki).

Techniques: Expressing, Incubation, Recombinant, Western Blot, Infection, Transfection, Plasmid Preparation, Immunostaining, Mutagenesis, SDS Page

Journal: Developmental cell

Article Title: The bone microenvironment increases phenotypic plasticity of ER+ breast cancer cells

doi: 10.1016/j.devcel.2021.03.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: FGF2 (C-2) , Santa Cruz , sc-74412.

Techniques: Virus, Recombinant, Reverse Transcription, Transfection, Sequencing, Negative Control, Luciferase, Plasmid Preparation, Modification, Software

Journal: Cell reports

Article Title: NLRP3 Cys126 palmitoylation by ZDHHC7 promotes inflammasome activation

doi: 10.1016/j.celrep.2024.114070

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pcDNA3-N-Flag-NLRP3 plasmid encoding mouse NLRP3 (Addgene plasmid # 75127) was a gift from Bruce Beutler. pcDNA3-Myc-ASC plasmid encoding human ASC (Addgene plasmid # 73952) and NLRP3-GFP plasmid (Addgene plasmid # 73955) were gifts from Christian Stehlik., Plasmid encoding human HA-ASC was obtained from GenScript.

Techniques: Recombinant, Control, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, Western Blot, Expressing, Mutagenesis, Transgenic Assay, Plasmid Preparation, Construct, Software, Microscopy

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Phosphorylation regulates FOXC2 function in vivo. (A) Endothelial cell-specific gain-of-function models for the analysis of FOXC2 phosphorylation. (B) Both models express comparable levels of the transgene, as evidenced by RT-PCR analysis of the indicated mRNAs from E15.5 lungs. Transgene expression was initiated at E13.5. (C) Macroscopic appearances of FOXC2ecGOF, pmFOXC2ecGOF, and control E15.5 embryos. (D and E) FOXC2 overexpression does not affect capillary sprouting. (F) Overexpression of FOXC2 but not pmFOXC2 promotes vascular remodeling in maturing capillaries. Note the increased capillary branching and density in FOXC2ecGOF embryos. Whole-mount staining of E15.5 head skin for pan-endothelial marker CD31 (green) and the transgene (red). The transgene expression was detected using anti-Myc antibody. Scale bars: 100 μm (D), 38 μm (E), 35 μm (F). (G) Quantification of vascular branching, density, and sprouting at the vascular front in the control, pmFOXC2ecGOF, and FOXC2ecGOF embryos. n = 3 per genotype. *, P < 0.05. n.s., nonsignificant.

Article Snippet: The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and either immunoblotted with sheep anti-human FOXC2 antibody (R&D Systems) or subjected to autoradiography.

Techniques: Phospho-proteomics, In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Over Expression, Staining, Marker

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Analysis of FOXC2 phosphorylation. (A) Endogenous and recombinant human FOXC2 are similarly phosphorylated in primary LECs and immortalized cell lines. Cell lysates were treated (+) or not treated (−) with lambda protein phosphatase (λ-PPase) and analyzed by Western blotting with anti-FOXC2 or anti-Myc antibodies. (B) Schematic representation of FOXC2 phosphorylation sites. FHD, forkhead domain; TA, transactivation domains (5, 34); PD, phosphorylated domain. Phosphorylation sites identified by LC-MS/MS are shaded in red; phosphorylation sites identified by mutagenesis are shaded in yellow. Peptides detected by MS in tryptic and Glu-C digests are underlined in cyan and green, respectively. Amino acid numbering is the same as in the endogenous protein (NP_005242). (C) Substitution of eight phosphorylation sites in Myc-FOXC2 with alanine abolishes the phosphorylation-dependent electrophoretic mobility shift. Lysates of cells transfected with a plasmid expressing the phosphorylation-deficient mutant Myc-pmFOXC2 were treated (+) or not treated (−) with λ-PPase and analyzed by Western blotting with anti-Myc antibody. (D) FOXC2 phosphorylation-deficient mutant (pm) has increased electrophoretic mobility compared to the wild-type (wt) protein. Shown is Western blot analysis of lysates from HepG2 cells transduced with adenoviruses expressing Myc-FOXC2 or Myc-pmFOXC2.

Article Snippet: The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and either immunoblotted with sheep anti-human FOXC2 antibody (R&D Systems) or subjected to autoradiography.

Techniques: Phospho-proteomics, Recombinant, Western Blot, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Electrophoretic Mobility Shift Assay, Transfection, Plasmid Preparation, Expressing, Transduction

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: FOXC2 interacts with peptidyl-prolyl cis/trans isomerase PIN1, alpha isoform of the regulatory subunit B of the protein phosphatase PP2A (PPP2R2A), and ERK1/2 protein kinases. (A) Coimmunoprecipitation assays with anti-Myc antibody demonstrate the association of Myc-FOXC2 with endogenous PIN1 in HeLa cells transduced with recombinant Ad-Myc-FOXC2. Shown is Western blot (WB) of anti-Myc immunoprecipitates consecutively probed with anti-Myc and anti-PIN1 antibodies. Control immunoprecipitation was performed from extracts of HeLa cells transduced with recombinant adenovirus expressing bacterial β-galactosidase (Ad-LacZ). (B) Myc-FOXC2 binds to endogenous PPP2R2A and ERK1/2 in HepG2 cells transduced with recombinant Ad-Myc-FOXC2. Coimmunoprecipitation assays were performed and analyzed as in A, except that anti-PPP2R2A and anti-total ERK1/2 antibodies were used for immunoblotting. (C) Immunocomplex kinase assays demonstrate that Myc-FOXC2 is phosphorylated in vitro by the coprecipitating endogenous ERK1/2 kinases. Shown is Western blot (WB) of anti-Myc immunoprecipitates incubated in the presence of [γ-32P]ATP and phosphorimage of the corresponding membrane. The blot was consecutively probed with anti-Myc, anti-total ERK1/2 and anti-active ERK1/2 (p-ERK1/2) antibodies. The identity of ERK1/2 was confirmed by immunocomplex kinase assays with anti-Myc antibody using lysates of HepG2 cells stimulated with PMA in the presence or absence of 10 mM U0126, a selective inhibitor of upstream MEK. (D) Inhibition of ERK1/2 does not modify the electrophoretic mobility of endogenous FOXC2 in LECs. Shown is a Western blot of total LEC lysates consecutively probed with anti-FOXC2 and anti-active ERK1/2 antibodies. (E) The electrophoretic mobility of endogenous FOXC2 changes after release from serum starvation-induced cell cycle arrest in LECs, suggesting CDK involvement in FOXC2 phosphorylation.

Article Snippet: The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and either immunoblotted with sheep anti-human FOXC2 antibody (R&D Systems) or subjected to autoradiography.

Techniques: Transduction, Recombinant, Western Blot, Control, Immunoprecipitation, Expressing, In Vitro, Incubation, Membrane, Inhibition, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Phosphorylation regulates FOXC2-mediated transcription in primary LECs. (A) Immunofluorescent staining for Myc (green), lymphatic marker PROX1 (red), and DNA (blue) of LECs transduced with adenoviruses expressing wild-type Myc-FOXC2, phosphorylation-deficient mutant Myc-pmFOXC2, or control bacterial β-galactosidase (LacZ). Note that wild-type and mutant FOXC2 have similar expression levels and subcellular localization. Bars, 20 μm. (B) Phosphorylation regulates FOXC2 transcriptional activity. Gene expression profiling was performed on the adenovirus-transduced LECs shown in panel A. Genes whose expression changed >2-fold in response to the loss of FOXC2 phosphorylation (FDR < 0.01) are shown in orange (upregulated) and purple (downregulated) in the Volcano plot of significance against the fold change in gene expression. Vertical dotted lines mark the 2-fold change limits. (C) RT-PCR validation of the gene expression profiling results. Genes upregulated or downregulated >2-fold in response to the loss of FOXC2 phosphorylation are shown in orange and purple, respectively; genes affected <2-fold are shown in gray. No change in FOXC2 expression reflects equally efficient cell transduction with Ad-Myc-FOXC2 and Ad-Myc-pmFOXC2. The data are presented as log2-transformed fold change in gene expression normalized to a housekeeping gene (GAPDH). Horizontal dotted lines mark the 2-fold change limits. Shown are the means and standard deviations of triplicate determinations in a single experiment representative of two independent experiments. (D) Heat map representation of the differences in gene expression in response to the loss of FOXC2 phosphorylation. The left heat map shows expression levels of 57 of 59 genes downregulated >2-fold (FDR < 0.01) in Ad-Myc-pmFOXC2-transduced LECs compared to Ad-Myc-FOXC2-transduced LECs. The right heat map shows expression levels of 57 out of 88 genes upregulated >2-fold (FDR < 0.01) in the same cells. Three biological replicates are shown for each condition. The color key at the lower left corresponds to the mean-centered, arctan-transformed log2-fold change in gene expression falling within the range from −π/2 to π/2. Blue denotes genes with relative decreased expression; red denotes genes with relative increased expression.

Article Snippet: The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and either immunoblotted with sheep anti-human FOXC2 antibody (R&D Systems) or subjected to autoradiography.

Techniques: Phospho-proteomics, Staining, Marker, Transduction, Expressing, Mutagenesis, Control, Activity Assay, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Transformation Assay

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Phosphorylation differentially regulates FOXC2 binding to genomic target sites in the context of native chromatin but not in vitro. We used genome-wide ChIP-chip to compare the binding of adenovirus-expressed wild-type Myc-FOXC2 and the phosphorylation-deficient mutant Myc-pmFOXC2 to physiological binding sites occupied by endogenous FOXC2 in primary LECs. Endogenous FOXC2 enrichment profiles are shown at the top of each panel. Purple peaks indicate FOXC2-enriched regions; their relative occupancies by Myc-FOXC2 and Myc-pmFOXC2 are shown in callout boxes in green and orange, respectively. Vertical axes represent MAT score. Binding sites are numbered as in Norrmén et al. (8); genomic coordinates refer to the hg18 human genome assembly. An unbound control region is shown in the lower right panel. The ChIP-chip results were validated by ChIP-qPCR with primers flanking ∼100-bp sequences within the FOXC2-enriched regions. The results are presented as the fold enrichment over the unbound control region. Green and orange bars correspond to wild-type Myc-FOXC2 and Myc-pmFOXC2, respectively. Shown are the means and standard deviations of triplicate determinations. An EMSA was used to compare the in vitro binding of adenovirus-expressed wild-type Myc-FOXC2, Myc-pmFOXC2, and deletion mutant Myc-FOXC2 D219-366 to naked dsDNA from the ChIP-enriched regions or the unbound control region. Binding specificity was controlled with adenovirus-expressed bacterial β-galactosidase (LacZ) and anti-Myc antibody. Asterisks indicate the positions of the antibody-supershifted complexes.

Article Snippet: The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and either immunoblotted with sheep anti-human FOXC2 antibody (R&D Systems) or subjected to autoradiography.

Techniques: Phospho-proteomics, Binding Assay, In Vitro, Genome Wide, ChIP-chip, Mutagenesis, Control, ChIP-qPCR

Journal: Neuron

Article Title: Human cortical neurogenesis is altered via glucocorticoid-mediated regulation of ZBTB16 expression.

doi: 10.1016/j.neuron.2024.02.005

Figure Lengend Snippet: Figure 2. Glucocorticoids alter the expression profile of ZBTB16 (A) RT-qPCR results for ZBTB16 and MAP2 across hCO development. (B) Western blots of ZBTB16 and ACTIN across hCO development. Each lane contains protein from a pool of three hCOs. (C) Representative images of day 30 baseline hCOs stained for DCX, SOX2, MAP2, ZBTB16, and DAPI. (D) Western blots of ZBTB16 and ACTIN in hCOs treated with 100 nM dex at day 43 and analyzed 7 days later at day 50. Each lane contains protein from a pool of three hCOs, and the six replicates were generated in two independent hCO batches. (E) Quantification of the effect of 100 nM dex over 7 days on ZBTB16 protein expression in day 50 hCOs normalized over ACTIN. (F) Quantification of the effect of 100 nM dex over 7 days on ZBTB16 mRNA levels normalized over endogenous genes and day 40 baseline ZBTB16 expression levels. RT-qPCR, quantitative reverse-transcription polymerase chain reaction; hCOs, human cerebral organoids; Veh, vehicle; Dex, dexamethasone. For (E), signifi- cance was tested with two-tailed Mann-Whitney comparison between treatment and vehicle. For (F), significance was tested with one-way ANOVA with Ben- jamini, Krieger, and Yekutieli multiple testing correction (p = 0.0003). Box and whisker plots represent 25th to 75th percentile of the data with the center line representing the median and whiskers representing minima and maxima. Mann-Whitney p values for (E) or post hoc p values for (F): ****p % 0.0001, ***p % 0.001, **p % 0.01, *p % 0.05, ns p > 0.05. Scale bars, 50 mm. See also Figure S3.

Article Snippet: More specifically, the human ZBTB16 ORF (NM_006006.5, 2034 bp) sequence was amplified from a plasmid delivered fromGenScript and the F2A-GFP from the Snap25-LSL-2A-GFP vector (Addgene, #61575).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Generated, Reverse Transcription, Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY, Comparison, Whisker Assay

Journal: Neuron

Article Title: Human cortical neurogenesis is altered via glucocorticoid-mediated regulation of ZBTB16 expression.

doi: 10.1016/j.neuron.2024.02.005

Figure Lengend Snippet: Figure 4. ZBTB16 is necessary for the effects of glucocorticoids on PAX6+EOMES+ basal progenitors (A) Treatment and analysis workflow in hCOs derived from edited No.1 iPSCs with either ZBTB16+/+ or ZBTB16+/ genotypes. (B) Western blots for ZBTB16 and ACTIN in ZBTB16+/+- or ZBTB16+/-derived hCOs at veh and dex. Each lane contains protein from a pool of three organoids. (C) Quantification of western blot results. (D) Representative images of FCa of ZBTB16+/+-derived hCOs per treatment condition. TBR2 is an alternative name for EOMES. (E) Quantification of the FCa results. (F) Representative images of FCa of ZBTB16+/-derived hCOs per treatment condition. (G) Quantification of the FCa results. DMSO, dimethyl sulfoxide; hCOs, human cerebral organoids; Veh, vehicle; Dex, dexamethasone; FCa, flow cytometry analysis. For (C), (E), and (G), significance was tested with two-way ANOVA with Benjamini, Krieger, and Yekutieli multiple testing correction (C: p.interaction = 0.03, E: p.interaction = 0.0068, G: p.interaction = 0.97). Data are represented as mean ± SEM. Post hoc p values: **p % 0.01, ns p > 0.05. See also Figure S5 and Table S5.

Article Snippet: More specifically, the human ZBTB16 ORF (NM_006006.5, 2034 bp) sequence was amplified from a plasmid delivered fromGenScript and the F2A-GFP from the Snap25-LSL-2A-GFP vector (Addgene, #61575).

Techniques: Derivative Assay, Western Blot, Cytometry

Journal: Cell

Article Title: Divergent sensory pathways of sneezing and coughing

doi: 10.1016/j.cell.2024.08.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After staining, sections were mounted using Fluoromount-G and imaged using a Nikon C2 confocal system.

Techniques: Virus, Plasmid Preparation, Recombinant, Labeling, Digital PCR, Avidin-Biotin Assay, Lysis, Reverse Transcription, RNAscope, Multiplex Assay, Mutagenesis, TaqMan Assay, Software, Real-time Polymerase Chain Reaction

Journal: Circulation Research

Article Title: Foxc2 Is a Common Mediator of Insulin and Transforming Growth Factor β Signaling to Regulate Plasminogen Activator Inhibitor Type I Gene Expression

doi: 10.1161/01.res.0000207407.51752.3c

Figure Lengend Snippet: Figure 1. Foxc2 transactivates the human PAI-1 promoter. A, Dose-dependent transactivation of the PAI-1 promoter by Foxc2 in BAECs. B, Luciferase assay using BAECs with different PAI-1 reporters. Note that luciferase activities by caFoxc2 are more than 60-fold. C, Luciferase assay using 3T3-L1 with Foxc expression vectors with or without a dominant-negative form of Foxc2, dnFoxc2. **P0.005, *P0.05 vs control. N9 from 3 experiments.

Article Snippet: The membranes were incubated with 3% nonfat dry milk at RT for 1 h and then probed with anti-β-actin antibody (Sigma) or anti-FOXC2 antibody (Santa Cruz).

Techniques: Luciferase, Expressing, Dominant Negative Mutation, Control

Journal: Circulation Research

Article Title: Foxc2 Is a Common Mediator of Insulin and Transforming Growth Factor β Signaling to Regulate Plasminogen Activator Inhibitor Type I Gene Expression

doi: 10.1161/01.res.0000207407.51752.3c

Figure Lengend Snippet: Figure 2. Foxc proteins bind the PAI-1 promoter. A, Specific Fox-binding sites in the human PAI-1 promoter. FBE is located adjacent to 1 of the TRSs, and IRE is between the 2 Sp1 sites. Both IRE and FBE are highly conserved between human and mouse. Forkhead consensus sequences are shown in bold. Shown below are the corresponding sites of primers used for electrophoretic mobility- shift and ChIP assays. B, Electrophoretic mobility-shift assay for FBE (Lanes 1 to 13) and IRE (lanes 21 to 33). Labeled probes for FBE, IRE, and their mutants were incubated with in vitro–translated Fox proteins in the absence or presence of cold competitors as indicated at the top. In vitro synthesized product from the pcDNA3 was used as negative con- trol (NC). Arrowheads indicate specific DNA–protein complexes. C, Luciferase assay using BAECs with mutated report- ers for the FBE and IRE. Mutations of both sites significantly attenuated lucif- erase activity by Foxc2. *P0.05 vs con- trol. N9 from 3 experiments.

Article Snippet: The membranes were incubated with 3% nonfat dry milk at RT for 1 h and then probed with anti-β-actin antibody (Sigma) or anti-FOXC2 antibody (Santa Cruz).

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Incubation, In Vitro, Synthesized, Luciferase, Activity Assay

Journal: Circulation Research

Article Title: Foxc2 Is a Common Mediator of Insulin and Transforming Growth Factor β Signaling to Regulate Plasminogen Activator Inhibitor Type I Gene Expression

doi: 10.1161/01.res.0000207407.51752.3c

Figure Lengend Snippet: Figure 3. Foxc2 functionally competes with FOXO1 to regulate PAI-1 gene expression via IRE. A, Foxc2-induced luciferase activity was inhibited by FOXO1 in BAECs. **P0.01 vs control. N9 from 3 experiments. B through D, Insulin treatment. BAECs (B) and 3T3-L1 cells (C) were examined by luciferase assay using Fox expression vectors and pGLuc85 reporter with or without insulin treatment. D, BAECs were transfected with mutated reporters. *P0.05, significant difference between the presence and absence of insulin treat- ment. N9 from 3 experiments.

Article Snippet: The membranes were incubated with 3% nonfat dry milk at RT for 1 h and then probed with anti-β-actin antibody (Sigma) or anti-FOXC2 antibody (Santa Cruz).

Techniques: Gene Expression, Luciferase, Activity Assay, Control, Expressing, Transfection

Journal: Circulation Research

Article Title: Foxc2 Is a Common Mediator of Insulin and Transforming Growth Factor β Signaling to Regulate Plasminogen Activator Inhibitor Type I Gene Expression

doi: 10.1161/01.res.0000207407.51752.3c

Figure Lengend Snippet: Figure 4. Foxc2 functionally interacts with Smad3/4 to regulate PAI-1 transcription. A, Luciferase assay using BAECs with Foxc and Smad expression vectors along with pGLuc884 reporter. N12 from 4 experiments. B, GST pull-down assay. [35S]- labeled Foxc1 or Foxc2 were incubated with GST alone (lane 1) or GST-Smad fusion proteins (lanes 2 to 4). Ten percent input of [35S]-labeled proteins was shown on the left. Coomassie blue- stained SDS-PAGE at the bottom showed levels of GST fusion proteins. C, Luciferase assay using BAECs with Smad3 and Smad4 along with Foxc2 and/or FOXO1. *P0.05, **P0.01 vs control. N9 from 3 experiments.

Article Snippet: The membranes were incubated with 3% nonfat dry milk at RT for 1 h and then probed with anti-β-actin antibody (Sigma) or anti-FOXC2 antibody (Santa Cruz).

Techniques: Luciferase, Expressing, Pull Down Assay, Labeling, Incubation, Staining, SDS Page, Control

Journal: Circulation Research

Article Title: Foxc2 Is a Common Mediator of Insulin and Transforming Growth Factor β Signaling to Regulate Plasminogen Activator Inhibitor Type I Gene Expression

doi: 10.1161/01.res.0000207407.51752.3c

Figure Lengend Snippet: Figure 5. Foxc2 regulates endogenous expression of PAI-1. A, ChIP assay. HMEC-1 cells (1106) were cross-linked and subjected to ChIP using PBS (No Ab), anti-Smad4, anti-FOXC2, and anti- FOXO1 antibodies, followed by immuno- precipitation. The immunoprecipitated DNA was analyzed by PCR using primer sets for hFBE (left) or hIRE (right). B, Upregulation of endogenous PAI-1 expression by Foxc2 in response to insu- lin and TGF-1. MEECs were transfected with Foxc2 expression vector or pcDNA3 (Mock) and treated with either 10 g/mL of insulin for 18 hours or 10 ng/mL of TGF-1 for 4 hours. Total RNA was sub- jected to semiquantitative RT-PCR analysis.

Article Snippet: The membranes were incubated with 3% nonfat dry milk at RT for 1 h and then probed with anti-β-actin antibody (Sigma) or anti-FOXC2 antibody (Santa Cruz).

Techniques: Expressing, Immunoprecipitation, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Circulation Research

Article Title: Foxc2 Is a Common Mediator of Insulin and Transforming Growth Factor β Signaling to Regulate Plasminogen Activator Inhibitor Type I Gene Expression

doi: 10.1161/01.res.0000207407.51752.3c

Figure Lengend Snippet: Figure 6. TGF-1–driven PAI-1 expres- sion is impaired in Foxc2/ mice. A, Relative mRNA abundance of Foxc2 in adult tissues of wild-type and Foxc2/ mice in response to TGF-1. The CT Values for untreated wild-type mice were used as control, and bars represent rela- tive mRNA abundance compared with untreated wild-type mice in each tissue. ANOVA analysis is indicated at the top. Foxc2 transcripts were less abundant in untreated Foxc2/ mice than in untreated wild type (WT), consistent with Foxc2 haploinsufficiency. B, Relative mRNA abundance of PAI-1 in adult tis- sues of wild-type and Foxc2/ mice in response to TGF-1. The CT values for untreated wild-type mice were used as control, and bars represent relative mRNA abundance compared with untreated wild-type mice in each tissue. MeanSEM of 2CT from 5 mice are shown.

Article Snippet: The membranes were incubated with 3% nonfat dry milk at RT for 1 h and then probed with anti-β-actin antibody (Sigma) or anti-FOXC2 antibody (Santa Cruz).

Techniques: Control