Journal: Cells

Article Title: A Personalized 14-3-3 Disease-Targeting Workflow Yields Repositioning Drug Candidates

doi: 10.3390/cells14080559

Figure Lengend Snippet: Primers used to generate 14-3-3γ, 3xHA-TH, and GFP-SLP76 mutants.

Article Snippet: To generate GFP-SLP76 construct, the human SLP76 sequence (SinoBiological, HG11237-M) was amplified by PCR and cloned in frame into the EcoRI/BamHI sites of pEGFP-N1 [ ].

Techniques:

Journal: Cells

Article Title: A Personalized 14-3-3 Disease-Targeting Workflow Yields Repositioning Drug Candidates

doi: 10.3390/cells14080559

Figure Lengend Snippet: Mutants R57C, R57G, R132C, and Y133S lose interactions with partner phosphoprotein in vitro and in cells. ( A ) A novel in vitro BRET assay developed in this study to assess the interaction between recombinant 14-3-3γ (C-terminally tagged with nanoluc (nLuc; BRET donor)) and partner phosphopeptide (N-terminally tagged with FITC; BRET acceptor). Upon the addition of furimazine, substrate for nLuc, light emitted by nLuc excites FITC in the close proximity (<10 nm), which occurs when FITC-phosphopeptide bound to 14-3-3γ-nLuc. ( B ) The production and purification of recombinant 14-3-3γ-nLuc from E. coli showed similar expression levels and purity between wild-type and mutants 14-3-3γ-nLuc. ( C – E ) Recombinant 14-3-3γ-nLuc (1 nM) was mixed with increasing doses of FITC-pTH ( C ), FITC-ppLRRK2 ( D ), or FITC-pSLP76 ( E ) before the addition of 250 nM furimazine. Wild-type 14-3-3γ-nLuc exhibits increased BRET signal in a dose-dependent manner, while mutants 14-3-3γ-nLuc show significantly decreased BRET signals indicating their incapability to efficiently bind FITC-phosphopeptides. ( F – H ) Cellular interactions of 14-3-3γ with TH or SLP76 as determined by IP. N2a cells were co-transfected with wild-type or R57C mutant 14-3-3γ-GFP and 3xHA-TH. IP of 14-3-3γ was performed using a nanobody against GFP, and the co-precipitation of TH was analyzed by SDS-PAGE and Western blot. Antibody against GFP was used for the detection of 14-3-3γ and against HA for TH ( F ). N2a cells were co-transfected with GFP-SLP76 and wild-type or R57C mutant 14-3-3γ-3xHA. IP of SLP76 was performed using a nanobody against GFP, and the co-precipitation of 14-3-3γ was analyzed by SDS-PAGE and Western blot. Antibody against GFP was used for the detection of SLP76 and against HA for 14-3-3γ ( G ). Quantification of the coimmunoprecipitation between 14-3-3γ and TH or SLP76 ( H ). Data in the panels ( C – E , H ) represent mean ± SEM ( n ≥ 3). Statistical analysis for panel H was performed using one sample t -test, **** p < 0.0001.

Article Snippet: To generate GFP-SLP76 construct, the human SLP76 sequence (SinoBiological, HG11237-M) was amplified by PCR and cloned in frame into the EcoRI/BamHI sites of pEGFP-N1 [ ].

Techniques: In Vitro, Bioluminescence Resonance Energy Transfer, Recombinant, Purification, Expressing, Transfection, Mutagenesis, SDS Page, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: An Informatics-assisted Label-free Approach for Personalized Tissue Membrane Proteomics: Case Study on Colorectal Cancer *

doi: 10.1074/mcp.M110.003087

Figure Lengend Snippet: IHC analysis of STOML2 expression in CRC tissues. Representative IHC staining patterns of STOML2 are presented. ( A–C ) A , Expression of STOML2 in a CRC specimens containing both tumor and nontumor cells. STOML2 was strongly expressed in tumor cells but not in neighboring nontumor epithelium cells. The boxed area for nontumor (b) and tumor (c) cells were enlarged and shown in ( B ) and ( C ), respectively. D , Membrane localization of STOML2 overexpressed in tumor cells. The boxed area in ( D ) was enlarged and shown in ( E ). F , STOML2 expression in tissue sections containing benign epithelium cells. ( G–J ) Intense STOML2 expression in tumor cells of CRC specimens from patients with Dukes' stage A - D (intensity 3+ and staining cell percentage 100%). scale bar: 500 μm in ( A ); 50 μm in ( B and C ); 100 μm in ( D , F–J ); or 5 μm in ( E ).

Article Snippet: Expression and purification of the recombinant protein were confirmed with immunoblotting using a mouse monoclonal antibody against the STOML2 protein (ProteinTech Group Inc., Chicago, IL).

Techniques: Expressing, Immunohistochemistry, Membrane, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: An Informatics-assisted Label-free Approach for Personalized Tissue Membrane Proteomics: Case Study on Colorectal Cancer *

doi: 10.1074/mcp.M110.003087

Figure Lengend Snippet: Expression of STOML2 in normal, benign and malignant colorectal tissues

Article Snippet: Expression and purification of the recombinant protein were confirmed with immunoblotting using a mouse monoclonal antibody against the STOML2 protein (ProteinTech Group Inc., Chicago, IL).

Techniques: Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: An Informatics-assisted Label-free Approach for Personalized Tissue Membrane Proteomics: Case Study on Colorectal Cancer *

doi: 10.1074/mcp.M110.003087

Figure Lengend Snippet: Association between expression levels of STOML2 in tissue and clinicopathologic characteristics of 205 CRC patients

Article Snippet: Expression and purification of the recombinant protein were confirmed with immunoblotting using a mouse monoclonal antibody against the STOML2 protein (ProteinTech Group Inc., Chicago, IL).

Techniques: Expressing, Concentration Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: An Informatics-assisted Label-free Approach for Personalized Tissue Membrane Proteomics: Case Study on Colorectal Cancer *

doi: 10.1074/mcp.M110.003087

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of the 5-year overall survival in CRC

Article Snippet: Expression and purification of the recombinant protein were confirmed with immunoblotting using a mouse monoclonal antibody against the STOML2 protein (ProteinTech Group Inc., Chicago, IL).

Techniques: Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: An Informatics-assisted Label-free Approach for Personalized Tissue Membrane Proteomics: Case Study on Colorectal Cancer *

doi: 10.1074/mcp.M110.003087

Figure Lengend Snippet: A , ELISA of the plasma levels of STOML2 in 70 CRC patients and 70 age-matched healthy controls. B , ROC curves of CEA and STOML2 based on the ELISA data shown in ( A ). C , Comparison of STOML2 and CEA plasma levels in early- and advanced-stage CRC patients. D , ELISA analysis of STOML2 expression in plasma from CRC patients of different stages (early stage: TNM stage I and II, n = 29; advanced stage: TNM stage III and IV, n = 41) and from healthy controls ( n = 70). *, p < 0.01, **, p < 0.001; Student's t test.

Article Snippet: Expression and purification of the recombinant protein were confirmed with immunoblotting using a mouse monoclonal antibody against the STOML2 protein (ProteinTech Group Inc., Chicago, IL).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Comparison, Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: An Informatics-assisted Label-free Approach for Personalized Tissue Membrane Proteomics: Case Study on Colorectal Cancer *

doi: 10.1074/mcp.M110.003087

Figure Lengend Snippet: Detection sensitivity of CEA and STOML2 as CRC biomarkers

Article Snippet: Expression and purification of the recombinant protein were confirmed with immunoblotting using a mouse monoclonal antibody against the STOML2 protein (ProteinTech Group Inc., Chicago, IL).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury

doi: 10.1084/jem.20111493

Figure Lengend Snippet: Deficiency in ADAP or SLP-76 attenuates neutrophil recruitment and protects mice from AKI. (A and B) Irradiated WT mice were reconstituted with BM from WT ( n = 4), Slp76 −/− ( n = 4), Slp76 Y112/128 (DM; n = 4), Slp76 Y145 (SM; n = 4), and ADAP −/− mice ( n = 4). 6–8 wk later, mice were subjected to RIR or sham injury, and the number of neutrophils recruited into the kidney (A) and plasma creatinine were assessed 24 h later (B). (C) Representative H&E staining of kidney outer medulla from chimeric mice was assessed 24 h after sham operation or renal IRI. Insets show a twofold magnified image. Bars, 50 µm. (D and E) Chimeric mice were pretreated with an IgG control antibody or a blocking anti–E-selectin antibody (Ab) 10 min after sham or renal IRI. 24 h later, the number of neutrophils in the kidney (D) and creatinine levels in the plasma (E) were determined. Results are presented as mean ± SEM. # , P < 0.05.

Article Snippet: In brief, BM cells were isolated from gene-deficient mice ( Slp76 −/− ) and were subjected to lineage depletion by using a lineage cell depletion kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Irradiation, Clinical Proteomics, Staining, Control, Blocking Assay

Journal: The Journal of Experimental Medicine

Article Title: Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury

doi: 10.1084/jem.20111493

Figure Lengend Snippet: Leukocyte rolling and adhesion in kidney or cremaster venules after ischemia-reperfusion is dependent on SLP-76 and ADAP. (A and B) Untreated LysM-GFP mice or LysM-GFP mice pretreated with different antibodies (anti–Mac-1 antibody [M1-70], n = 3; anti–LFA-1 antibody [TIB217], n = 3; and anti–E-selectin antibody [9A9], n = 3) were subjected to RIR, and the rolling velocity (A) and the number of adherent leukocytes (B) in venules of the kidney were determined. (C) Representative pictures of cremaster muscle postcapillary venules of untreated LysM-GFP mice and LysM-GFP mice pretreated with a blocking anti–E-selectin antibody (Ab) 4 h after renal IRI. Bars, 25 µm. (D and E) 2 h after RIR, WT mice were injected i.v. with fluorescently labeled BM cells from WT ( n = 3), Slp76 −/− ( n = 3), Slp76 Y145 (SM; n = 3), Slp76 Y112/128 (DM; n = 3) or ADAP −/− ( n = 3) mice. 2 h later, leukocyte rolling velocity (D) and the number of adherent cells (E) in venules of the kidney were determined. (F and G) The cremaster muscle of WT ( n = 3), Slp76 −/− ( n = 3), Slp76 Y145 ( n = 3), Slp76 Y112/128 ( n = 3), and ADAP −/− ( n = 3) mice was subjected to ischemia (30 min)/reperfusion (120 min) injury, and mean rolling velocity (F) and the number of adherent cells (G) in postcapillary venules of the cremaster muscle were determined. Results are presented as means ± SEM. # , P < 0.05.

Article Snippet: In brief, BM cells were isolated from gene-deficient mice ( Slp76 −/− ) and were subjected to lineage depletion by using a lineage cell depletion kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Blocking Assay, Injection, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury

doi: 10.1084/jem.20111493

Figure Lengend Snippet: SLP-76 tyrosines are required for E-selectin–mediated slow leukocyte rolling and Gα i -independent adhesion. (A–C) The carotid artery of chimeric mice reconstituted with BM from WT ( n = 3) or Slp76 −/− ( n = 3) mice (A), with WT ( n = 3), SM ( n = 3), or DM ( n = 3) mice (B), or with WT ( n = 3) or ADAP ( n = 3) mice (C) was cannulated with a catheter, which was connected to autoperfused flow chambers. Mean rolling velocity of neutrophils on E-selectin (left) and E-selectin and ICAM-1 (right) is presented as means ± SEM. The wall shear stress in all flow chamber experiments was 5–6 dyn/cm 2 . # , P < 0.05. (D–F) Mixed chimeric mice were generated by injecting BM cells from LysM-GFP + WT (WT; D–F) mice and Slp76 −/− ( Slp76 −/− ; D), SM (E) and DM mice (E), or ADAP −/− mice ( ADAP −/− ; F) into lethally irradiated WT mice. Cumulative histogram of rolling velocities of 100 GFP + (WT) and 100 GFP − leukocytes in inflamed cremaster muscle venules of mixed chimeric mice ( n = 4) treated with PTx and a monoclonal blocking P-selectin antibody (RB40.34). The insets show the mean rolling velocity ± SEM. # , P < 0.05. (G–I) Numbers of adherent cells per square millimeter in murine cremaster muscle venules. The cremaster muscle was exteriorized 2 h after intrascrotal injection of 500 ng TNF or after injection of TNF and PTx in chimeric mice reconstituted with BM from WT mice (G–I; n = 3) or Slp76 −/− (G; n = 3), SM and DM (H), or ADAP −/− (I) mice. Results are presented as means ± SEM. # , P < 0.05. (J) Mixed chimeric mice were generated by injecting retrovirally transduced hematopoietic stem cells (Slp-76–WT construct, WT-c.; Slp76-R448K construct, R448-c.; empty vector, no-c.) from Slp76 −/− mice into lethally irradiated WT mice. Cumulative histogram of rolling velocities of transduced (WT-c., n = 100; R448-c., n = 100; no-c., n = 100) leukocytes in inflamed cremaster muscle venules of mixed chimeric mice ( n = 3) treated with PTx and a monoclonal blocking P-selectin antibody. The inset shows the mean rolling velocity ± SEM. # , P < 0.05. (K) Coimmunoprecipitation of SLP-76 and ADAP. BM-derived WT neutrophils were plated on uncoated (unstimulated) or E-selectin–coated wells for 10 min, and then lysates were prepared followed by immunoprecipitation (IP) with ADAP antibody. Precipitates were immunoblotted (IB) with antibodies to total SLP-76 (top) and total ADAP (bottom).

Article Snippet: In brief, BM cells were isolated from gene-deficient mice ( Slp76 −/− ) and were subjected to lineage depletion by using a lineage cell depletion kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Shear, Generated, Irradiation, Blocking Assay, Injection, Construct, Plasmid Preparation, Derivative Assay, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury

doi: 10.1084/jem.20111493

Figure Lengend Snippet: E-selectin modulates integrin adhesiveness by regulating integrin affinity and avidity. (A and B) HL-60 cells were transfected with control shRNA or with shRNAs against SLP-76 or ADAP and subjected to immunoblot to verify down-regulation of SLP-76 (A) and ADAP down-regulation (B). Total p38 MAPK expression was used as loading control. (C) HL-60 cells were transfected with control shRNA or with shRNAs against SLP-76 or ADAP and were used in a flow chamber adhesion assay. Flow chambers were coated with E-selectin and a control IgG antibody (open bars) or KIM127 antibody, recognizing the intermediate affinity conformation of LFA-1 (closed bars) and perfused with transfected HL-60 cells. Bars represent the number of adherent cells per field of view as mean ± SEM of three independent experiments. # , P < 0.05. (D) Flow chamber adhesion assay with human leukocytes in whole blood pretreated with DMSO or specific inhibitors against PLC (U73122), PI3Kγ, or Btk (LFM-A13). Flow chambers were coated with E-selectin and a control IgG antibody (open bars) or KIM127 antibody (closed bars). Bars show the number of adherent cells per field of view as mean ± SEM of three independent experiments. # , P < 0.05. (E) Chimeric mice reconstituted with BM from WT, Slp76 Y112/128 , ADAP −/− , Btk −/− , Pik3cg −/− , or Plcg2 −/− mice were used to investigate LFA-1 clustering on rolling cells in vivo. Mice were pretreated with TNF and PTx 2 h before the experiments, and a blocking P-selectin antibody and an Alexa Fluor–conjugated LFA-1 antibody were injected immediately before preparing the cremaster muscle for intravital microscopy analysis. Cells were classified as clustered if fluorescence was >1.5 times increased at one edge of the cell. Data are shown as percentage of clustered cells as mean ± SEM of three independent measurements with at least 100 cells per group. # , P < 0.05; *, P < 0.05 versus other groups. (F) Representative images of rolling leukocytes in inflamed cremaster venules of WT and Btk −/− mice stained for LFA-1 clustering. Bars, 10 µm.

Article Snippet: In brief, BM cells were isolated from gene-deficient mice ( Slp76 −/− ) and were subjected to lineage depletion by using a lineage cell depletion kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Transfection, Control, shRNA, Western Blot, Expressing, Cell Adhesion Assay, In Vivo, Blocking Assay, Injection, Intravital Microscopy, Fluorescence, Staining

Journal: The Journal of Experimental Medicine

Article Title: Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury

doi: 10.1084/jem.20111493

Figure Lengend Snippet: Gα i -independent leukocyte extravasation and neutrophil recruitment is defective in Slp76 −/− , Slp76 Y112/128 , and ADAP −/− mice. (A) Numbers of extravasated leukocytes in cremasteric venules of TNF–treated chimeric mice reconstituted with BM from WT ( n = 4), Slp76 −/− ( n = 4), Slp76 Y145 (SM; n = 4), Slp76 Y112/128 (DM; n = 4), or ADAP −/− mice ( n = 4) per 1.5 × 10 4 –µm 2 tissue area. The measurements were performed 2 h after intrascrotal TNF injection. The same groups were also analyzed after pretreatment with 4 µg PTx i.v. (+PTx; WT mice + PTx, n = 4; Slp76 −/− mice + PTx, n = 4; Slp76 Y145 mice + PTx, n = 4; Slp76 Y112/128 mice + PTx, n = 4; and ADAP −/− mice + PTx, n = 4). (B) Neutrophil influx into the peritoneal cavity 8 h after 1-ml injection of 3% thioglycollate into chimeric mice reconstituted with BM from WT ( n = 5), Slp76 −/− ( n = 4), Slp76 Y112/128 ( n = 4), or ADAP −/− mice ( n = 4). The same groups were also analyzed after pretreatment with 4 µg PTx i.v. (WT mice + PTx, n = 4; Slp76 −/− mice + PTx, n = 4; Slp76 Y112/128 mice + PTx, n = 4; and ADAP −/− mice + PTx, n = 4). Total numbers of neutrophils in the peritoneal lavage fluid were determined by flow cytometry and hemocytometer count. Results are presented as means ± SEM. # , P < 0.05.

Article Snippet: In brief, BM cells were isolated from gene-deficient mice ( Slp76 −/− ) and were subjected to lineage depletion by using a lineage cell depletion kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Injection, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury

doi: 10.1084/jem.20111493

Figure Lengend Snippet: SLP-76 is required for the activation of Btk, PLCγ2, PI3Kγ, and p38 MAPK after E-selectin engagement. BM-derived neutrophils were plated on uncoated (unstimulated) or E-selectin–coated wells for 10 min, and then lysates were prepared. (A and B) Lysates of WT, Tyrobp −/− Fcrg −/− (A; n = 3), and Btk −/− (B; n = 3) were immunoprecipitated (IP) with a SLP-76 antibody followed by immunoblotting (IB) with a general phosphotyrosine (PY; 4G10) antibody or total SLP-76 antibody. (C) Lysates of WT and Slp76 −/− neutrophils were immunoprecipitated with an Syk ( n = 3) or Btk antibody ( n = 3) followed by immunoblotting with a general phosphotyrosine (4G10) antibody, total Syk antibody ( n = 3), or total Btk antibody ( n = 3). Lysates were immunoblotted with antibody to phosphorylated PLCγ2 (p-PLCγ2 [Tyr1217]; n = 3), total PLCγ2 ( n = 3), phosphorylated Akt ( n = 3), total Akt ( n = 3), phosphorylated p38 MAPK (p-p38), or total p38 MAPK ( n = 3). (D) Lysates of WT and Slp76 Y112/128 neutrophils were immunoprecipitated with Btk antibody ( n = 3) followed by immunoblotting with a general phosphotyrosine (4G10) antibody or total Btk antibody ( n = 3). Lysates were immunoblotted with antibody to phosphorylated PLCγ2 (Tyr1217; n = 3), total PLCγ2 ( n = 3), phosphorylated Akt ( n = 3), total Akt ( n = 3), phosphorylated p38 MAPK, or total p38 MAPK ( n = 3). (E and F) Lysates of WT, Tyrobp −/− Fcrg −/− (E; n = 3), and Btk −/− neutrophils (F; n = 3) were immunoprecipitated with an ADAP antibody followed by immunoblotting with a general phosphotyrosine (4G10) antibody or total ADAP antibody. (G) Lysates of WT and ADAP −/− neutrophils were immunoprecipitated with an Syk ( n = 3) or Btk antibody ( n = 3) followed by immunoblotting with a general phosphotyrosine (4G10) antibody, total Syk antibody ( n = 3), or total Btk antibody ( n = 3). Lysates were immunoblotted with antibody to phosphorylated PLCγ2 (Tyr1217; n = 3), total PLCγ2 ( n = 3), phosphorylated Akt ( n = 3), total Akt ( n = 3), phosphorylated p38 MAPK, or total p38 MAPK ( n = 3). (H) Lysates of WT and Slp76 Y145 neutrophils were immunoprecipitated with Btk antibody followed by immunoblotting with a general phosphotyrosine antibody (4G10). Lysates were also immunoblotted with antibody to phosphorylated PLCγ2 (Tyr1217; n = 3), total PLCγ2 ( n = 3), phosphorylated Akt ( n = 3), total Akt ( n = 3), phosphorylated p38 MAPK, or total p38 MAPK ( n = 3).

Article Snippet: In brief, BM cells were isolated from gene-deficient mice ( Slp76 −/− ) and were subjected to lineage depletion by using a lineage cell depletion kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Activation Assay, Derivative Assay, Immunoprecipitation, Western Blot