Western Blot:Article Title: Ring finger protein 213 regulates B-cell receptor signaling, metabolism, and development in B lymphocytes
Article Snippet: Subsequently, cell lysis was carried out with RIPA lysis buffer (Beyotime, P0013B). .. The cell lysates were examined via SDS-PAGE, and immunoblotting was performed with the following antibodies: anti-pSYK (Cell Signaling Technology, CST, 2710S), anti-SYK (CST, 13198S), anti-pCD19 (CST, 3571S), anti-CD19 (CST, 90176S), anti-β-actin (Proteintech, 66009-1-Ig), anti-pNFκB pP65 (CST, 3033S), anti-NFκB P65 (CST, 4764S), anti-pSTAT1 (CST, 9167S), anti-STAT1 (CST, 14994S), anti-pSTAT3 (CST, 9145S), anti-STAT3 (CST, 9139S), anti-pSTAT5 (CST, 4322S), anti-STAT5 (CST, 94205S), anti-pPI3K pP85/pP55 (CST, 4228 L), anti-PI3K P85 (CST, 4292S), anti-pAKT (CST, 4060 L), anti-AKT (CST, 9272S), anti-pmTOR (CST, 5536S), anti-mTOR (CST, 2983S), anti-pS6 (CST, 4858 L), anti-S6 (CST, 2217S), anti-pFOXO1 (CST, 9461S), anti-FOXO1 (CST, 2880S), anti-CTCF (ABclonal, A19588), anti-SPIB (CST, 14337S), anti-ETS1 (ZenBio, R382074), anti-GABPA (HuaBio, HA500187 ), anti-FLI1 (HuaBio, HA500149 ), anti-PU.1 (ABclonal, A20461), anti-GAPDH (Proteintech, 60004-1-Ig), anti-PIK3C3 (ABclonal, A12295), anti-PTEN (CST, 9188S), anti-pSHIP-1 (CST, 3941S), SHIP-1 (ABclonal, A3571), anti-NOTCH2 (ABclonal, A24877), anti-pHSL (CST, 4126S), anti-HSL (CST, 4107S), anti-ATGL (CST, 2138S), anti-LC3 I/II (ABclonal, A5618), IκBα (ABclonal, A19714), pIκBα (SelleckChem, F2237), pIKKα/β (CST, 2697S), pP38 (SelleckChem, F0159), HSP90 (ABclonal, A5027), pJNK1/2 (ABclonal, AP0473), pERK1/2 (ABclonal, AP0472). ..
Article Title: A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2
Article Snippet: .. The cells were lysed and subjected to SDS-PAGE separation and western blotting analysis using anti-p-SYK (Cat# 2710T, Cell Signaling Technology), anti-SYK (Cat# 2712T, Cell Signaling Technology), anti-p-BLNK (Cat# 3601T, Cell Signaling Technology), anti-BLNK (Cat# 36438T, Cell Signaling Technology) and anti-β-ACTIN (Cat# A2228, Sigma-Aldrich) antibodies. (C) Schematic illustration of the extraction of GAGs with Titanium beads. (D) Toluidine blue gel analysis of heparin 10 μg, heparin extract 50 μg, plasma extract 0.75 mg and ROSA cells extract 0.75 mg. (E) Heparin or plasma extract was treated with heparinases (Hps) I, II and III, chondroitinase ABC (ChABC) or in combination for 24 h or 48 h and were analyzed by 15% polyacrylamide gel electrophoresis and stained with toluidine blue. (F) Quantification of heparin in plasma from eight different healthy donors using Anti IIa Assay Kit. (G) Binding of BDCA-2 ECD to ELISA-plates coated with heparin (1 mg/mL) and under conditions where indicated concentrations of exogenous heparin (left panel), ROSA cell extract (middle panel) or plasma extract (right panel) were added. .. BDCA-2 ECD binding was detected with Strep-Tactin HRP conjugate (IBA).
Article Title: Trem2-dependent Insl3 regulation via Dap12-Syk-PI3K pathway: A new pathogenic mechanism in cryptorchidism.
Article Snippet: The target protein was visualized by using the Bio-Rad ChemiDoc MP imaging system. .. A variety of antibodies were utilized for WB, including anti-Trem2 (1: 3000, ab86491, Sheep monoclonal, Abcam), anti-Insl3 (1: 1000, ab199536, Rabbit monoclonal, Abcam), anti-Dap12 (1: 1000, ab283679, Rabbit monoclonal, Abcam), anti-Syk (1: 2000, 13198, Rabbit monoclonal, Cell Signaling Technology), antiPI3K (1: 2000, ab302958, Rabbit monoclonal, Abcam), anti-GAPDH (1: 2000, 60004–1-1G, Mouse polyclonal, Proteintech, Rosemont, IL, USA), anti-IgG (ab172730, Rabbit monoclonal, Abcam), anti-rabbitHRP conjugated (1: 5000, SA00001–2, Proteintech), anti-mouse-HRP conjugated (1: 5000, SA00001, Proteintech), and anti-sheep-HRP conjugated (1: 4000, SC-2473, Santa Cruz Biotechnology) antibodies. .. For IP, anti-Dap12 (2 μg, ab283679, Rabbit monoclonal, Abcam) or anti-Syk (1: 50, 13198, Rabbit monoclonal, Cell Signaling Technology) antibodies were utilized in the study.
SDS Page:Article Title: A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2
Article Snippet: .. The cells were lysed and subjected to SDS-PAGE separation and western blotting analysis using anti-p-SYK (Cat# 2710T, Cell Signaling Technology), anti-SYK (Cat# 2712T, Cell Signaling Technology), anti-p-BLNK (Cat# 3601T, Cell Signaling Technology), anti-BLNK (Cat# 36438T, Cell Signaling Technology) and anti-β-ACTIN (Cat# A2228, Sigma-Aldrich) antibodies. (C) Schematic illustration of the extraction of GAGs with Titanium beads. (D) Toluidine blue gel analysis of heparin 10 μg, heparin extract 50 μg, plasma extract 0.75 mg and ROSA cells extract 0.75 mg. (E) Heparin or plasma extract was treated with heparinases (Hps) I, II and III, chondroitinase ABC (ChABC) or in combination for 24 h or 48 h and were analyzed by 15% polyacrylamide gel electrophoresis and stained with toluidine blue. (F) Quantification of heparin in plasma from eight different healthy donors using Anti IIa Assay Kit. (G) Binding of BDCA-2 ECD to ELISA-plates coated with heparin (1 mg/mL) and under conditions where indicated concentrations of exogenous heparin (left panel), ROSA cell extract (middle panel) or plasma extract (right panel) were added. .. BDCA-2 ECD binding was detected with Strep-Tactin HRP conjugate (IBA).
Extraction:Article Title: A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2
Article Snippet: .. The cells were lysed and subjected to SDS-PAGE separation and western blotting analysis using anti-p-SYK (Cat# 2710T, Cell Signaling Technology), anti-SYK (Cat# 2712T, Cell Signaling Technology), anti-p-BLNK (Cat# 3601T, Cell Signaling Technology), anti-BLNK (Cat# 36438T, Cell Signaling Technology) and anti-β-ACTIN (Cat# A2228, Sigma-Aldrich) antibodies. (C) Schematic illustration of the extraction of GAGs with Titanium beads. (D) Toluidine blue gel analysis of heparin 10 μg, heparin extract 50 μg, plasma extract 0.75 mg and ROSA cells extract 0.75 mg. (E) Heparin or plasma extract was treated with heparinases (Hps) I, II and III, chondroitinase ABC (ChABC) or in combination for 24 h or 48 h and were analyzed by 15% polyacrylamide gel electrophoresis and stained with toluidine blue. (F) Quantification of heparin in plasma from eight different healthy donors using Anti IIa Assay Kit. (G) Binding of BDCA-2 ECD to ELISA-plates coated with heparin (1 mg/mL) and under conditions where indicated concentrations of exogenous heparin (left panel), ROSA cell extract (middle panel) or plasma extract (right panel) were added. .. BDCA-2 ECD binding was detected with Strep-Tactin HRP conjugate (IBA).
Clinical Proteomics:Article Title: A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2
Article Snippet: .. The cells were lysed and subjected to SDS-PAGE separation and western blotting analysis using anti-p-SYK (Cat# 2710T, Cell Signaling Technology), anti-SYK (Cat# 2712T, Cell Signaling Technology), anti-p-BLNK (Cat# 3601T, Cell Signaling Technology), anti-BLNK (Cat# 36438T, Cell Signaling Technology) and anti-β-ACTIN (Cat# A2228, Sigma-Aldrich) antibodies. (C) Schematic illustration of the extraction of GAGs with Titanium beads. (D) Toluidine blue gel analysis of heparin 10 μg, heparin extract 50 μg, plasma extract 0.75 mg and ROSA cells extract 0.75 mg. (E) Heparin or plasma extract was treated with heparinases (Hps) I, II and III, chondroitinase ABC (ChABC) or in combination for 24 h or 48 h and were analyzed by 15% polyacrylamide gel electrophoresis and stained with toluidine blue. (F) Quantification of heparin in plasma from eight different healthy donors using Anti IIa Assay Kit. (G) Binding of BDCA-2 ECD to ELISA-plates coated with heparin (1 mg/mL) and under conditions where indicated concentrations of exogenous heparin (left panel), ROSA cell extract (middle panel) or plasma extract (right panel) were added. .. BDCA-2 ECD binding was detected with Strep-Tactin HRP conjugate (IBA).
Polyacrylamide Gel Electrophoresis:Article Title: A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2
Article Snippet: .. The cells were lysed and subjected to SDS-PAGE separation and western blotting analysis using anti-p-SYK (Cat# 2710T, Cell Signaling Technology), anti-SYK (Cat# 2712T, Cell Signaling Technology), anti-p-BLNK (Cat# 3601T, Cell Signaling Technology), anti-BLNK (Cat# 36438T, Cell Signaling Technology) and anti-β-ACTIN (Cat# A2228, Sigma-Aldrich) antibodies. (C) Schematic illustration of the extraction of GAGs with Titanium beads. (D) Toluidine blue gel analysis of heparin 10 μg, heparin extract 50 μg, plasma extract 0.75 mg and ROSA cells extract 0.75 mg. (E) Heparin or plasma extract was treated with heparinases (Hps) I, II and III, chondroitinase ABC (ChABC) or in combination for 24 h or 48 h and were analyzed by 15% polyacrylamide gel electrophoresis and stained with toluidine blue. (F) Quantification of heparin in plasma from eight different healthy donors using Anti IIa Assay Kit. (G) Binding of BDCA-2 ECD to ELISA-plates coated with heparin (1 mg/mL) and under conditions where indicated concentrations of exogenous heparin (left panel), ROSA cell extract (middle panel) or plasma extract (right panel) were added. .. BDCA-2 ECD binding was detected with Strep-Tactin HRP conjugate (IBA).
Staining:Article Title: A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2
Article Snippet: .. The cells were lysed and subjected to SDS-PAGE separation and western blotting analysis using anti-p-SYK (Cat# 2710T, Cell Signaling Technology), anti-SYK (Cat# 2712T, Cell Signaling Technology), anti-p-BLNK (Cat# 3601T, Cell Signaling Technology), anti-BLNK (Cat# 36438T, Cell Signaling Technology) and anti-β-ACTIN (Cat# A2228, Sigma-Aldrich) antibodies. (C) Schematic illustration of the extraction of GAGs with Titanium beads. (D) Toluidine blue gel analysis of heparin 10 μg, heparin extract 50 μg, plasma extract 0.75 mg and ROSA cells extract 0.75 mg. (E) Heparin or plasma extract was treated with heparinases (Hps) I, II and III, chondroitinase ABC (ChABC) or in combination for 24 h or 48 h and were analyzed by 15% polyacrylamide gel electrophoresis and stained with toluidine blue. (F) Quantification of heparin in plasma from eight different healthy donors using Anti IIa Assay Kit. (G) Binding of BDCA-2 ECD to ELISA-plates coated with heparin (1 mg/mL) and under conditions where indicated concentrations of exogenous heparin (left panel), ROSA cell extract (middle panel) or plasma extract (right panel) were added. .. BDCA-2 ECD binding was detected with Strep-Tactin HRP conjugate (IBA).
Binding Assay:Article Title: A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2
Article Snippet: .. The cells were lysed and subjected to SDS-PAGE separation and western blotting analysis using anti-p-SYK (Cat# 2710T, Cell Signaling Technology), anti-SYK (Cat# 2712T, Cell Signaling Technology), anti-p-BLNK (Cat# 3601T, Cell Signaling Technology), anti-BLNK (Cat# 36438T, Cell Signaling Technology) and anti-β-ACTIN (Cat# A2228, Sigma-Aldrich) antibodies. (C) Schematic illustration of the extraction of GAGs with Titanium beads. (D) Toluidine blue gel analysis of heparin 10 μg, heparin extract 50 μg, plasma extract 0.75 mg and ROSA cells extract 0.75 mg. (E) Heparin or plasma extract was treated with heparinases (Hps) I, II and III, chondroitinase ABC (ChABC) or in combination for 24 h or 48 h and were analyzed by 15% polyacrylamide gel electrophoresis and stained with toluidine blue. (F) Quantification of heparin in plasma from eight different healthy donors using Anti IIa Assay Kit. (G) Binding of BDCA-2 ECD to ELISA-plates coated with heparin (1 mg/mL) and under conditions where indicated concentrations of exogenous heparin (left panel), ROSA cell extract (middle panel) or plasma extract (right panel) were added. .. BDCA-2 ECD binding was detected with Strep-Tactin HRP conjugate (IBA).
Enzyme-linked Immunosorbent Assay:Article Title: A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2
Article Snippet: .. The cells were lysed and subjected to SDS-PAGE separation and western blotting analysis using anti-p-SYK (Cat# 2710T, Cell Signaling Technology), anti-SYK (Cat# 2712T, Cell Signaling Technology), anti-p-BLNK (Cat# 3601T, Cell Signaling Technology), anti-BLNK (Cat# 36438T, Cell Signaling Technology) and anti-β-ACTIN (Cat# A2228, Sigma-Aldrich) antibodies. (C) Schematic illustration of the extraction of GAGs with Titanium beads. (D) Toluidine blue gel analysis of heparin 10 μg, heparin extract 50 μg, plasma extract 0.75 mg and ROSA cells extract 0.75 mg. (E) Heparin or plasma extract was treated with heparinases (Hps) I, II and III, chondroitinase ABC (ChABC) or in combination for 24 h or 48 h and were analyzed by 15% polyacrylamide gel electrophoresis and stained with toluidine blue. (F) Quantification of heparin in plasma from eight different healthy donors using Anti IIa Assay Kit. (G) Binding of BDCA-2 ECD to ELISA-plates coated with heparin (1 mg/mL) and under conditions where indicated concentrations of exogenous heparin (left panel), ROSA cell extract (middle panel) or plasma extract (right panel) were added. .. BDCA-2 ECD binding was detected with Strep-Tactin HRP conjugate (IBA).
Incubation:Article Title: Trem2-dependent Insl3 regulation via Dap12-Syk-PI3K pathway: A new pathogenic mechanism in cryptorchidism.
Article Snippet: .. Primary antibody incubation was performed at 4 ◦C overnight: anti-BrdU (3 μg/mL, ab6326, Rat monoclonal, Abcam, Cambridge, MA, USA), anti-Insl3 (1: 1000, ab227974, Rabbit monoclonal, Abcam), anti-Trem2 (1: 500, ab305103, Rabbit monoclonal, Abcam), anti-Dap12 (1: 2000, ab283679, Rabbit monoclonal, Abcam), anti-Syk (1: 500, 13198S, Rabbit monoclonal, Cell Signaling Technology, Danvers, MA, USA), anti-IgG (ab172730, Rabbit monoclonal, Abcam), anti-IgG2a kappa (14–4321-82, Rat monoclonal, Thermo Fisher Scientific). .. Then, sections were exposed to enzymelabeled Goat anti-rabbit IgG complex derived from an IHC kit (Zhong Shan Golden Bridge Biological Technology, Beijing, China) or Goat antirat HRP-conjugated secondary antibody (1: 250, SC-2006, Santa Cruz Biotechnology, CA, USA) for 30 min after PBS rinsing.
other:Article Title: Thymoquinone Inhibits BTK Expression and Phosphorylation in B-Cell Lymphoma: A Novel Plant-Derived Therapeutic Approach
Article Snippet: The antibodies used in this work, were as follows: anti-phospho-BTK (pY551) (1:1,500) anti-BTK (1:1,500) from BD Pharmingen; anti-phospho-BTK (pY551) (1:1,500) from RD® Systems; anti-BTK (1:1,500) from Merck, Anti-Actin (1:1,500), Anti-GAPDH (1:1,000), Anti-BLNK (1:1,500), Anti-SYK (1:1,500), anti-phospho-BTK (pY223) and Anti-NF-KB (1:2,000) from Cell Signaling; Anti-Akt (1:2,000) from Invitrogen; Anti-14-3-3 ζ (1:2,000), IgG Anti-Rabbit (1:20,000) and IgG Anti-Mouse (1:20,000) were obtained from Santa Cruz Biotechnology.
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