wild type shp2 expressing plasmid pj3  (Addgene inc)

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Comparison of non-structural protein 1 sequences among closely related flaviviruses. ( A ): Ribbon model highlighting regions of the NS1 protein containing segments exposed at the outer surface to the host environment. ( B ): Sequence comparison showing regions with high sequence disparity. Amino acids depicted in red differ from the corresponding ZIKV NS1 amino acids. A represents positions with two sequences with amino acids identical to ZIKV NS1. The boxes highlight highly conserved sequences, amino acids 117–119 and 227–229, that were mutated to alanine in immunodominant regions 2 and 3.

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Comparison, Sequencing

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Western blot of NS1 mutants. ( A ): The blot was probed with anti-His6 antibody targeted toward the protein N-terminus. ( B ): The blot was probed with anti-ZIKV NS1 monoclonal antibody targeted toward the C-terminus. Uncropped gels are displayed in .

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Western Blot

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Ratios of mutant vs. wild-type binding of NS1 by patient samples. The blue line at 1 indicates equal binding. Numbers over 1 indicate increased binding to the named mutant, while under 1 indicate increased wild-type binding. ( A ): Ratios of mutant/wild-type binding from IgG in samples show preferential binding to 117–119 mutant NS1. ( B ): IgM shows preferential binding of mutant NS1 proteins in samples from the Dominican Republic. Colombia = Colombia samples from suspected ZIKV infection. Dom Rep = Dominican Republic samples from suspected ZIKV infection. 117–119 = ZIKV NS1 W117A, G118A, K119A. 227–229 = ZIKV NS1 H227A, T228A, L229A Comparisons were made using Kruskal–Wallis ANOVA. Asterisks represent significant comparisons (**** p < 0.0001; ** p < 0.01). The full dataset is presented in .

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Mutagenesis, Binding Assay, Infection

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Serum from goats immunized with ZIKV mutants show differential binding to NS1 proteins. Antibody-capture ELISA was performed on a mixture of serum from two goats immunized with ZIKV NS1 protein. Binding of WT ZIKV NS1 showed lower binding to WT ( A ), both mutants ( B , C ), as well as DENV2 WT NS1 ( D ). Data represent samples run in duplicate. Error bars (standard error of the mean) were smaller than the symbols as drawn. The experiment was repeated a total of three times. The legend in panel ( D ) also applies to panels ( A – C ).

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Protein Binding

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Process for purifying and cross-adsorbing ZIKV NS1 antibodies. ZIKV NS1 antibodies were affinity purified using a gravity flow column. ( A ): The bound and eluted fraction was put through a column with DENV NS1 twice. The unbound and eluted (non-DENV reactive) fraction is the cross-adsorbed, ZIKV-specific fraction. ( B ): Antibodies flowing through the column initially bind ZIKV at low signal strength. Following ZIKV NS1 column elution, strength of binding of the solution goes up, but DENV NS1 reactivity is present. Upon cross-adsorption against DENV NS1, specificity of the pAb solution goes up, as well as its binding avidity to ZIKV NS1.

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Affinity Purification, Binding Assay, Adsorption

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: ZIKV NS1 antigen-capture ELISA limit of detection and dynamic range. ( A ): ZIKV rNS1 shows binding significantly higher than BSA down to 7.8 ng/mL. The limit of detection of the assay is between 7.8 and 1.95 ng/mL NS1. ( B ): Linear regression of NS1 detection via ELISA demonstrates assay can quantify protein across a range of values. Comparisons were made using two-way ANOVA with a Holm–Sidak multiple comparisons test. Asterisks represent significant comparisons (**** p < 0.0001; * p < 0.05). Error bars represent the standard error of the mean.

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: Production of flavivirus NS1. Coomassie-stained SDS-PAGE images of induced (I) and uninduced (U) cultures from NS1 proteins of yellow fever virus ( A ), West Nile virus ( A , B ) and St. Louis Encephalitis virus ( B ). Bands indicate proteins similar to DENV and ZIKV NS1 WT production. Results from three different colonies [ , , ] for each flavivirus NS1 are indicated. Uncropped gels are displayed in .

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Staining, SDS Page, Virus

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: The ZIKV NS1 antigen-capture assay demonstrates low cross-reactivity to related viruses. Recombinant NS1 antigens were produced and assayed. A: Even at low levels of NS1 detection, the assay is specific to ZIKV NS1. Comparisons made using two-way ANOVA with a Holm–Sidak multiple comparisons test. Asterisks represent significant comparisons between ZIKV NS1 and all other NS1 proteins (**** p < 0.0001; * p < 0.05). Error bars represent the standard error of the mean.

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Recombinant, Produced

Journal: Viruses

Article Title: Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

doi: 10.3390/v13091771

Figure Lengend Snippet: ZIKV NS1 antigen-capture ELISA results from patients infected during the 2015–2016 outbreaks in Colombia and the Dominican Republic 1 .

Article Snippet: Antiserum against ZIKV rNS1 proteins was generated via immunization of goats with wild-type NS1 and NS1 with alanine substitution mutations of amino acids 117–119 or 227–229 were each inoculated in goats. (ProSci, San Diego, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Infection

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: SHP2 inhibits CRC cell proliferation and migration. ( A ) SHP2 knockdown by siSHP2#1 and #2 markedly increased the proliferation of HCT116 and SW480 cells. ( B ) Colony formation assays were conducted to estimate the growth rate of HCT116 and SW480 cells. SHP2 knockdown increased the colony numbers compared with the control group. Representative pictures of colonies (left) and quantification of colony numbers (right) are shown. ( C ) Transwell assay was performed to access the effect of SHP2 on cell migration by siRNA mediated knockdown. Representative pictures of cells (left) and quantification of cell numbers (right) are shown. ( D ) SW480 cells were transfected with pJ-SHP2 plasmid to overexpress SHP2 and the cell proliferation was accessed by MTT assay. ( E ) Colony formation assay was done by overexpression of SHP2 in SW480 cells. SHP2 overexpression decreased the colony numbers compared with the control group. Representative pictures of colonies (left) and quantification of colony numbers (right) are shown. ( F ) Transwell assay was performed to access the effect of SHP2 on cell migration by overexpressing SHP2 in SW480 cells. Representative pictures of cells (left) and quantification of cell numbers (right) are shown. Overexpression of SHP2 in HCT116 and SW480 cells rescued SHP2 knockdown induced cell proliferation ( G ), colony formation ( H ) and cell migration ( I ). ( J and K ) PHPS1 improved HCT116 and SW480 cell proliferation during 3 days in a time- and dose-dependent manner. ( L ) Cells were treated with PHPS1, and colony formation was measured after two weeks by crystal violet staining. PHPS1 increased the number of CRC cell colonies. (M)Transwell assay showing blockade of SHP2 phosphatase activity by PHPS1 improved CRC cell migratory ability. NC, non-silencing control siRNA. Values represent mean ± SEM (n = 3–4), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Migration, Knockdown, Control, Transwell Assay, Transfection, Plasmid Preparation, MTT Assay, Colony Assay, Over Expression, Staining, Activity Assay

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: SHP2 suppressing role in CRC is mediated by STAT3 dephosphorylation. ( A ) STAT3 phosphorylation was increased in CRC cells knocked down for SHP2 by siRNA#1 and #2. ( B ) SHP2 overexpression reduced STAT3 phosphorylation in SW480. ( C ) PHPS1 enhanced the levels of pSTAT3 in 10 mg/L time-dependently. ( D ) Nuclear distribution of pSTAT3 was enhanced after SHP2 knockdown (×400). ( E ) Cryptotanshinone significantly reversed CRC cell proliferation induced by PHPS1. Values represent mean ± SEM (n = 3), **P ≤ 0.01, compared with Vehicle group. ( F ) SW480 was more sensitive to IL-6-induced proliferation after SHP2 knockdown, which was rescued by SHP2 overexpression; meanwhile overexpression SHP2 inhibited IL-6 induced SW480 proliferation. Values represent mean ± SEM (n = 3) # P ≤ 0.05, **P ≤ 0.01, compared with NC + pJ3 group. Whole blots are shown in Supplementary Fig. .

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: De-Phosphorylation Assay, Phospho-proteomics, Over Expression, Knockdown

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Association between the combination of SHP2/nuclear STAT3 and survival in patients with CRC. ( A ) Kaplan–Meier analysis showed that Patients with high SHP2 and low nuclear STAT3 present better DSS (left panel) and DFS (right panel) than patients with low SHP2 and high nuclear STAT3. ( B ) Kaplan-Meier analysis showed that Patients with high SHP2 and low nuclear STAT3 present better DSS (left panel) and DFS (right panel) than patients with low SHP2 or high nuclear STAT3. ( C ) Kaplan-Meier analysis showed that patients with high SHP2 or low nuclear STAT3 present better DFS (right panel) but not DSS (left panel) than patients with low SHP2 and high nuclear STAT3. P values were determined using log-rank test.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques:

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Univariate Cox regression analysis for clinical parameters and the combinations of SHP2 and nuclear STAT3.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Adjuvant

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Multivariate Cox regression analysis for clinical parameters and the combinations of high SHP2 and low nuclear STAT3.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Adjuvant

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Multivariate Cox regression analysis of DFS for clinical parameters and the combinations of low SHP2 and high nuclear STAT3.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Adjuvant

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria

doi: 10.1038/s41598-017-00564-3

Figure Lengend Snippet: LPS treatment increases renal and podocyte Shp2 expression. ( a ) Immunoblots of Shp2 and tubulin in total kidney lysates. Control (saline, n = 6) and LPS-injected (n = 6) wild-type male mice were sacrificed 24 hours after injection. Representative images are shown and each lane represents an animal. Shp2 protein expression (left, normalized to tubulin) and mRNA (right, normalized to Tbp ) presented in bar charts as means + SEM. * p < 0.05 indicates a significant difference between saline and LPS-treated mice. ( b ) Co-immunostaining of Shp2 (green) and synaptopodin (Synpo, red) in kidney sections from saline and LPS-treated wild-type mice. Scale bar: 20 µm. ( c ) Differentiated E11 podocytes were treated with PBS for 24 hours and with LPS for 6, 12 and 24 hours. Cell lysates were immunoblotted for Shp2, synaptopodin, and actin. Protein expression was normalized to actin and presented in a bar chart as means + SEM from three independent experiments. * p < 0.05 and # p < 0.05 indicate a significant difference between PBS (24 h) and LPS-treated groups for Shp2 and synaptopodin, respectively. A.U., arbitrary units.

Article Snippet: E11 cells with Shp2 knockdown (KD) were rescued (KD-R) with Shp2 by transfection with pCMV human Shp2 wild type (Addgene), and stable cell lines were generated by neomycin (G418, 400 μg/ml) selection.

Techniques: Expressing, Western Blot, Control, Saline, Injection, Immunostaining

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria

doi: 10.1038/s41598-017-00564-3

Figure Lengend Snippet: Efficient and specific deletion of Shp2 in podocytes. ( a ) Genomic DNA was extracted from tissues (as indicated) of control (Ctrl) and pod-Shp2 knockout (KO) mice. Deletion of the floxed allele was detected by PCR, and GAPDH served as a loading control. ( b ) Immunoblots of Shp2 protein expression in isolated primary podocytes, adipose (epididymal fat), liver and muscle from Ctrl and KO mice. Tubulin and synaptopodin (Synpo, for podocytes) presented as loading controls. ( c ) Immunostaining of Shp2 (green) and synaptopodin (red) in kidney sections from Ctrl and KO mice. Scale bar: 50 µm.

Article Snippet: E11 cells with Shp2 knockdown (KD) were rescued (KD-R) with Shp2 by transfection with pCMV human Shp2 wild type (Addgene), and stable cell lines were generated by neomycin (G418, 400 μg/ml) selection.

Techniques: Control, Knock-Out, Western Blot, Expressing, Isolation, Immunostaining

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria

doi: 10.1038/s41598-017-00564-3

Figure Lengend Snippet: Pod-Shp2 KO mice are more resistant than controls to LPS-induced renal injury. ( a ) Schematic of experimental timeline for LPS administration and mice sacrifice. Body weight ( b ), kidney weight ( c ), kidney/body weight ratio ( d ), urinary proteins concentration ( e ) and blood urea nitrogen ( f ) of control (Ctrl, n = 8) and pod-Shp2 knockout (KO, n = 8) mice without (saline) and with LPS treatment. * p < 0.05 and ** p < 0.01 indicate a significant difference between saline and LPS treatments; † p < 0.05 and †† p < 0.01 indicate a significant difference between Ctrl and KO mice. Data were presented as means + SEM. A.U., arbitrary units.

Article Snippet: E11 cells with Shp2 knockdown (KD) were rescued (KD-R) with Shp2 by transfection with pCMV human Shp2 wild type (Addgene), and stable cell lines were generated by neomycin (G418, 400 μg/ml) selection.

Techniques: Concentration Assay, Control, Knock-Out, Saline

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria

doi: 10.1038/s41598-017-00564-3

Figure Lengend Snippet: Attenuated LPS-induced inflammatory response in pod-Shp2 KO mice. Renal mRNA of Il - 1b , Il - 6 and Tnfa ( a ), and plasma concentrations of IL-1β, TNFα, INFγ and IL-12 p70 ( b ) in saline and LPS-treated control (Ctrl, n = 6) and pod-Shp2 knockout (KO, n = 6) mice. ( c ) Kidney lysates from Ctrl and KO mice without (saline) and with LPS treatment were immunoblotted for phosphorylated NF-κB p65, JNK, p38, peIF2α and their respective proteins, spliced XBP1 (sXBP1), and actin as a loading control. Representative images are shown. Bar charts represent pNF-κB p65/NF-κB p65, pJNK/JNK, pp38/p38, peIF2α/eIF2α and sXBP1/actin as means + SEM. For all bar charts, * p < 0.05 and ** p < 0.01 indicate a significant difference between saline and LPS treatments; † p < 0.05 and †† p < 0.01 indicate a significant difference between Ctrl and KO mice. A.U., arbitrary units.

Article Snippet: E11 cells with Shp2 knockdown (KD) were rescued (KD-R) with Shp2 by transfection with pCMV human Shp2 wild type (Addgene), and stable cell lines were generated by neomycin (G418, 400 μg/ml) selection.

Techniques: Clinical Proteomics, Saline, Control, Knock-Out

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria

doi: 10.1038/s41598-017-00564-3

Figure Lengend Snippet: Attenuated LPS-induced Shp2 and nephrin phosphorylation in pod-Shp2 KO mice. ( a ) Kidney lysates from control (Ctrl) and pod-Shp2 knockout (KO) mice without (saline) and with LPS treatment were immunoblotted for pShp2 (Tyr542), Shp2 and actin as a loading control. Representative images are shown and each lane represents an animal. Bar chart represents pShp2 (Tyr542)/actin as means + SEM (n = 6). * p < 0.05 indicates a significant difference between saline and LPS treatments; † p < 0.05 indicates a significant difference between Ctrl and KO mice. A.U., arbitrary units. ( b ) Immunostaining of pNephrin (Y1176/Y1193) in kidney sections from Ctrl and KO mice without (saline) and with LPS treatment. Lower panel includes enlarged images that are highlighted by white boxes in the upper panel. Scale bar: 50 µm.

Article Snippet: E11 cells with Shp2 knockdown (KD) were rescued (KD-R) with Shp2 by transfection with pCMV human Shp2 wild type (Addgene), and stable cell lines were generated by neomycin (G418, 400 μg/ml) selection.

Techniques: Phospho-proteomics, Control, Knock-Out, Saline, Immunostaining

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria

doi: 10.1038/s41598-017-00564-3

Figure Lengend Snippet: Shp2 deficiency in E11 podocytes attenuates LPS-induced inflammatory response and ER stress. Differentiated podocytes with Shp2 knockdown (KD) and rescue (KD-R) were treated with PBS and with LPS for 24 hours. Cell lysates were subjected to immunoblots with primary antibodies as indicated. Representative images are shown. Bar charts of pNF-κB p65/NF-κB p65, pJNK/JNK, pp38/p38, cleaved Caspase3/tubulin ( a ), pPERK/PERK, peIF2α/eIF2α, pIRE1α/IRE1α and sXBP1/tubulin ( b ), pNephrin/Nephrin and pShp2/tubulin ( c ) are presented as means + SEM from three independent experiments. * p < 0.05 and ** p < 0.01 indicate a significant difference between PBS and LPS treatments; † p < 0.05 and †† p < 0.01 indicate a significant difference between KD-R and KD podocytes. A.U., arbitrary units.

Article Snippet: E11 cells with Shp2 knockdown (KD) were rescued (KD-R) with Shp2 by transfection with pCMV human Shp2 wild type (Addgene), and stable cell lines were generated by neomycin (G418, 400 μg/ml) selection.

Techniques: Knockdown, Western Blot

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria

doi: 10.1038/s41598-017-00564-3

Figure Lengend Snippet: Decreased LPS-induced cell migration in Shp2 deficient podocytes. ( a ) Differentiated E11 podocytes with Shp2 knockdown (KD) and rescue (KD-R) were cultured with PBS and LPS for 48 hours after wound induction. Images were acquired before treatment (0 h) and at 24 and 48 h post wound. Scale bar: 200 µm. Cell numbers in the wound track were counted and presented in the bar chart ( b ) as means + SEM from four independent experiments. * p < 0.05 and ** p < 0.01 indicate a significant difference between PBS and LPS treatments; †† p < 0.01 indicates a significant difference between KD-R and KD podocytes.

Article Snippet: E11 cells with Shp2 knockdown (KD) were rescued (KD-R) with Shp2 by transfection with pCMV human Shp2 wild type (Addgene), and stable cell lines were generated by neomycin (G418, 400 μg/ml) selection.

Techniques: Migration, Knockdown, Cell Culture

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria

doi: 10.1038/s41598-017-00564-3

Figure Lengend Snippet: List of primers used to determine mRNA of Shp2 and pro-inflammatory cytokines.

Article Snippet: E11 cells with Shp2 knockdown (KD) were rescued (KD-R) with Shp2 by transfection with pCMV human Shp2 wild type (Addgene), and stable cell lines were generated by neomycin (G418, 400 μg/ml) selection.

Techniques:

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: RvD1 prevents LPS-induced SHP2 oxidation and its inactivation in the protection of AJ integrity. A. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in presence and absence of RvD1 (200 ng/ml) for 30 min and PTP activity was measured using PTP-specific phosphopeptide as a substrate. B & C. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in presence and absence of RvD1 (200 ng/ml) for the indicated time periods, cell extracts were prepared and equal amounts of protein from control and each treatment were immunoprecipitated with VE-cadherin or SHP2 antibodies and the immunocomplexes were analyzed by IB for the indicated proteins. D. All the conditions were same as in panel B except that after immunoprecipitation with SHP2 antibodies the immunocomplexes were assayed for SHP2 activity as described in panel A. E. Quiescent HUVECs that were treated with and without LPS (500 ng/ml) in the presence and absence of Allopurinol (50 μM) for 30 min were analyzed for SHP2 activity as described in panel D. F. All the conditions were same as in panel B except the cell extracts were immunoprecipitated with SHP2 antibodies and the immunocomplexes were immunoblotted for Cys sulphonate to measure SHP2 Cys oxidation and the blot was reprobed for total SHP2 levels. G. All the conditions were the same as in panel E except that the cell extracts were analyzed for SHP2 Cys oxidation as described in panel F and the blot was reprobed for total SHP2 levels. H. HUVECs were transiently transfected with empty vector (EV) or Myc-tagged recombinant SHP2 expression vector (WT and C459S mutant), grown to confluence, quiesced, treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and cell extracts were prepared. An equal amount of protein from control and each treatment was immunoprecipitated with Cysteine sulphonate antibody and the immunocomplexes were analyzed by IB for Myc to show SHP2 Cysteine oxidation. An equal amount of protein from control and each treatment was also analyzed by WB for Myc to show the overexpression of SHP2. I. HUVECs that were transfected with WT or mutant SHP2 expression vector and quiesced were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and analyzed for SHP2 activity as described in panel D.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Activity Assay, Phospho-proteomics, Control, Immunoprecipitation, Transfection, Plasmid Preparation, Recombinant, Expressing, Mutagenesis, Over Expression

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: Pharmacological inhibition of SHP2 blunts the capacity of RvD1 in the attenuation of LPS-induced Frk activation, α-catenin and VE-cadherin Tyr phosphorylation and AJ disruption. A. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with or without PHPS1 (10 μM), a potent inhibitor of SHP2, for 30 min, cell extracts were prepared and an equal amount of protein from control and each treatment was immunoprecipitated with pTyr or VE-cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins. The same cell extracts were also analyzed for the indicated protein total levels. B. All the conditions were the same as in panel A except that the quiescent HUVEC monolayer after the treatments was stained double immunofluorescently for α-catenin and VE-cadherin as described in Figure legend 2E. C & D. Quiescent HUVEC monolayer was treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with or without PHPS1 (10 μM) for 2 h or the indicated time periods and subjected to dextran flux (C) or TER (D) assays, respectively. The bar graphs represent Mean±SD values of three experiments. *, p<0.05 vs control; # , p<0.05 vs LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Disruption, Control, Immunoprecipitation, Staining

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: Both ALX/FPR2 and GPR32 mediate the protective effects of RvD1 on LPS-induced endothelial AJ disruption and its barrier dysfunction. A. Cell extracts of control and various time periods of LPS (500 ng/ml)-treated HUVECs were analyzed by WB for ALX/FPR2 and GPR32 levels using their specific antibodies. B-D. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) in combination with and without Boc2 (3 μM), ALX/FPR2 inhibitor, for 30 min and XO activity (B), ROS production (C) and SHP2 activity (D) were measured. E. All the conditions were the same as in panel B except that cell extracts were prepared, and equal amounts of protein from control and each treatment were immunoprecipitated with pTyr or VE-Cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same cell extracts were also analyzed by WB for the total levels of the indicated proteins. F & G. Quiescent HUVEC monolayer was treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) alone or in combination with and without Boc2 (3 μM) for 2 h or the indicated time periods and subjected to dextran flux (F) and TER (G) assays, respectively. H-J. Quiescent HUVECs were incubated with either control IgG or GPR32 IgG (10 μg/ml) alone or in combination with and without Boc2 (3 μM) for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min and XO activity (H), ROS production (I) and SHP2 activity (J) were measured. K. All the conditions were the same as in panel H except that cell extracts were prepared and equal amounts of protein from control and each treatment were immunoprecipitated with pTyr or VE-cadherin antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same cell extracts were also analyzed by WB for the total levels of the indicated proteins. L. The quiescent HUVEC monolayer that was incubated with either control IgG, GPR32 IgG (10 μg/ml), Boc2 (3 μM) alone or in combination for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 30 min was stained double immunofluorescently for α-catenin and VE-cadherin as described in Figure legend 2E. M & N. Quiescent HUVECs monolayer was incubated with either control IgG or GPR32 IgG (10 μg/ml) alone or in combination with and without Boc2 (3 μM) for 30 min followed by treatment with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for 2 h or the indicated time periods and subjected to dextran flux (M) and TER (N) assays, respectively. The bar graphs represent Mean±SD values of three experiments. *, p<0.05 vs control or control IgG; # , p<0.05 vs LPS or con IgG+LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Disruption, Control, Activity Assay, Immunoprecipitation, Incubation, Staining

Journal: Redox Biology

Article Title: Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function

doi: 10.1016/j.redox.2017.02.023

Figure Lengend Snippet: RvD1 attenuates LPS-induced aortic endothelial AJ disruption and hyper-permeability via blocking XO activity and SHP2 inactivation. A. C57BL/6 mice which were kept on chow diet were administered intraperitoneally with RvD1 (10 μg/kg body weight) every 2 days for 3 times before injecting LPS (5 mg/kg body weight) and 24 h later the aortas were isolated, tissue extracts were prepared and an equal amount of protein from each condition was analyzed for XO activity as described in Figure legend 4B. B. All the conditions were the same as in panel A except that tissue extracts containing an equal amount of protein from each condition were immunoprecipitated with Cys sulphonate antibodies and the immunocomplexes were analyzed by IB for SHP2. The same tissue extracts were analyzed by WB for total SHP2 levels. C. All the conditions were the same as in panel A except that tissue extracts were analyzed for SHP2 activity as described in Figure legend 5D. D & E. All the conditions were the same as in panel A except that tissue extracts were immunoprecipitated with pTyr (D) or VE-cadherin (E) antibodies and the immunocomplexes were analyzed by IB for the indicated proteins using their specific antibodies. The same tissue extracts were analyzed by WB for the indicated protein total levels. F. All the conditions were same as in panel A except that after isolation the aortas were opened longitudinally, fixed, permeabilized, blocked and co-immunostained for α-catenin and VE-cadherin as described in Figure legend 2E. G. All the conditions were the same as in panel A except that mice were anesthetized and 0.1 ml of 1% Evans Blue (EB) dye was injected into the tail vein. After 20 min, the blood vessels were perfused with PBS through the left ventriculum and the aortas were isolated and photographed. After taking the pictures, the aortas were minced, incubated in formaldehyde solution at 55 °C for 24 h, centrifuged and the optical density of the supernatant was measured at 610 nm in SpectraMax 190 spectrophotometer (Molecular Devices). The aortic endothelial barrier permeability was expressed as ng of EB dye extravasated per mg aorta. The bar graphs represent Mean±SD values of three experiments with 2 animals/group or 5 animals minimum. *, p<0.05 vs control; # , p<0.05 vs LPS.

Article Snippet: Wild type SHP2 (12283) and mutant SHP2 (C459S) (12284) plasmids were received from Addgene (Cambridge, MA) .

Techniques: Disruption, Permeability, Blocking Assay, Activity Assay, Isolation, Immunoprecipitation, Injection, Incubation, Spectrophotometry, Control

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. Inhibition of FGF signaling did not activate Src or FAK. BAEC were transduced with Ad-GFP or Ad-FGFR1DN. Cells in normal growth medium were lysed and total cell lysates were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. B , C , D , Quantitative analysis of pY416 Src (B), pY527 Src (C), pY397 Fak (D) shown in Fig. 3A. The value of NT, standardized with β-tubulin, was designated as 1. (n = 3). E , F . Reduced SHP2 expression in cells lacking FGF signaling. Western analysis using BAEC total cell lysates left untreated or transduced with either Ad-GFP or Ad-FGFR1DN. G , Quantitative analysis of SHP2 levels shown in Fig. 3F. The value of Ad-GFP at MOI 37.5, standardized with β-tubulin, was designated as 1. (n = 3 Mean ± SD, * P <0.05, by t-test compared with Ad-GFP, MOI 37.5). H , I . VE-cadherin-SHP2 interaction was disrupted by FGF signaling inhibition. BAEC were transduced with Ad-GFP or Ad-FGFR1DN. Cells were lysed and immunoprecipitated (IP) with anti-VE-cadherin (H) or anti-SHP2 (I) and subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. NT denotes no transduction. J . Catalytically inactive, dominant-negative SHP2 increased Y658 VE-cadherin phosphorylation. BAEC were transduced with lentivirus wild-typeSHP2 (WT) or dominant-negative-SHP2 (C/S). Cells were lysed and total cell lystes were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies.

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Inhibition, Transduction, SDS Page, Western Blot, Expressing, Immunoprecipitation, Dominant Negative Mutation, Phospho-proteomics

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. SHP2 mRNA levels were not decreased in endothelial cells lacking FGF signaling. Quantitative RT-PCR analysis of total RNA isolated from BAEC. Total RNA was isolated from BAEC transduced with Ad-GFP or Ad-FGFR1DN. SHP2 mRNA levels were measured with real-time PCR and normalized to GAPDH expression (Mean ± SD, * P <0.05, by t-test compared with NT). NT denotes no transduction. B . Western blotting of total cell lysates isolated from BAEC transduced with Ad-GFP or Ad-FGFR1DN and treated with 10 µg/ml cycloheximide for up to 36 hours. C . Quantitative analysis of SHP2 Western analysis described in B. The value at time point 0 was designated as 1. (n = 3 Mean ± SD, * P <0.05, by t-test compared with Ad-GFP). D . SHP2 is degraded via the lysosomal pathway in the absence of FGF signaling. Confluent BAEC transduced with either Ad-GFP or Ad-FGFR1DN were treated with 1 µM MG132, 20 µM lactacystin, 20 µM chloroquine or 25 mM NH 4 Cl for 24 hr. Total cell lysates were analyzed by Western blot. E . Quantitative analysis of SHP2 expression shown in Fig. 2D. The value of Ad-GFP control (DMSO) treatment, standardized with β-tubulin, was designated as 1. (n = 3, Mean ± SD, * P <0.05, by t-test compared with Ad-FGFR1DN control).

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Quantitative RT-PCR, Isolation, Transduction, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. Increased phosphorylation of VE-cadherin Y658 in cells lacking FGF signaling was restored to the basal level by SHP2 overexpression. BAEC were transduced with Ad-FGFR1DN and Ad-GFP or Ad-SHP2. Cells were lysed and total cell lysates were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. B . Quantitative analysis of SHP2 shown in Fig. 5A. The value of NT, standardized with β-tubulin, was designated as 1. (n = 3 Mean ± SD, * P <0.05, by t-test compared with NT, ** P <0.05, by t-test compared with Ad-FGFR1DN+Ad-GFP). C . SHP2 overexpression restored p120-catenin/VE-cadherin association. BAEC were transduced with Ad-FGFR1DN and Ad-GFP or Ad-SHP2. Total cell lysates were isolated and immunoprecipitated with VE-cadherin antibody. Immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies using the same membrane after stripping and reprobing. D , E , Immunostaining of quiescent and fully confluent BAEC transduced with Ad-FGFR1DN (D), or Ad-SHP2 and Ad-FGFR1DN (E). Cells were stained for VE-cadherin (green), SHP2 (red), and HA (FGFR1DN, blue). Arrows indicate gap formations between cells. F . Quantitative analysis of immunostaining evaluating the gap formation. Percent of gap area in each image was calculated using NIH Image J software using 6 different images. Data shown as mean ± SD *: P <0.05 by t-test. Scale Bars: 10 µm. G . SHP2 overexpression rescued the FGFR1DN effect on endothelial permeability. BAEC were transduced with Ad-SHP2 and Ad-FGFR1DN, and endothelial monolayer permeability was evaluated with the ECIS system. Transendothelial resistance was measured every 5 minutes for 17 hours after the onset of adenoviral transduction. NT denotes no transduction.

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Phospho-proteomics, Over Expression, Transduction, SDS Page, Western Blot, Isolation, Immunoprecipitation, Membrane, Stripping Membranes, Immunostaining, Staining, Software, Permeability

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

doi: 10.1371/journal.pone.0037600

Figure Lengend Snippet: A. Under normal circumstances, SHP2 is associated with VE-cadherin and dephosphorylates Y658 site of VE-cadherin, which results in p120-catenin coupling, enhancing VE-cadherin retention at adherens junctions. FGF signaling is required for VE-cadherin-SHP2 interaction. B . In cells expressing the FGFR1DN construct, SHP2 is downregulated and is not able to associate with VE-cadherin due to the lack of FGF signaling, which increases phosphorylation level of VE-cadherin Y658, leading to decoupling of p120-catenin; therefore, VE-cadherin stability at cell-cell junctions is impaired.

Article Snippet: Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech).

Techniques: Expressing, Construct, Phospho-proteomics