Journal: Medicina

Article Title: Meta-Analysis of Survival Effects of Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1)

doi: 10.3390/medicina58121867

Figure Lengend Snippet: Characteristics included for the study of ROR1.

Article Snippet: H. Chang (2015) [ ] , Republic of Korea , Gastric cancer , 424 , IHC , Rabbit polyclonal antibody (1:25; Abcam) , Staining > 50% , - , 0.8 (0.53–1.21) p = 0.189.

Techniques: Staining, Expressing, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: A thiazole-derived oridonin analogue exhibits antitumor activity by directly and allosterically inhibiting STAT3

doi: 10.1074/jbc.RA119.009801

Figure Lengend Snippet: CYD0618 disturbs the function of STAT3–SH2 domain via covalent modification of Cys-542. A and F, recombinant His–WT–STAT3 or His–C542S–STAT3 was treated with DMSO or CYD0618 for 1 h at room temperature. The mixtures were then incubated with the indicated purified FLAG kinases for 1 h at room temperature. Immunoblotting (IB) using the pTyr-705 STAT3 antibody reflects the effects of CYD0618 on kinase-induced phosphorylation of WT–STAT3 (A) or C542S–STAT (F) in vitro. B and C, CYD0618 inhibits the association of upstream kinases and STAT3. A2780 cells were pre-treated with CYD0618 (3 μm) for 2 h before stimulation with IL-6 (10 ng/ml) or EGF (20 ng/ml) for 30 min. Afterward, the whole-cell extracts were subjected to immunoprecipitation (IP) and immunoblotted with the indicated antibodies. D, C542S mutation regains the association of EGFR and STAT3. HeLa cells transfected with WT–STAT3 or C542S–STAT3 vector were pre-treated with CYD0618 for 2 h, followed by incubation of EGF (20 ng/ml) for 30 min. The whole-cell lysates were subjected to immunoprecipitation and immunoblotted with the indicated antibodies. E, effects of CYD0618 on the binding of Ac-pYLPQTV-NH2 to WT–STAT3 or C542S–STAT3 using the pulldown assay. Following incubation of CYD0618 for 1 h at room temperature, the recombinant His–WT–STAT3 or His–C542S–STAT3 was incubated with biotinylated Ac-pYLPQTV-NH2 (PPB) or biotinylated Ac-YLPQTV-NH2 (PB) and streptavidin beads for 1 h at room temperature. The mixtures were then subjected to immunoblotted with His antibody. G, representative global view of the GSH-mediated allosteric effect of STAT3 protein (PDB code 4E68). STAT3 protein alone is shown by the gray cartoon model; GSH–STAT3 complex is shown by the orange model. The disulfide bond between GSH and Cys-542 is identified and indicated by yellow line. H, representative view of the conformation change of the SH2 domain in STAT3 protein. The STAT3 protein alone is shown in gray, and GSH–STAT3 complex is shown in orange. The critical residues Lys-591, Arg-609, Ser-611, and Ser-613 are identified and indicated. The interaction between these two residues and pTyr-705 (stick model) of other STAT3 protein is also indicated.

Article Snippet: Antibodies Phospho-STAT1 (Tyr-701), phospho-STAT3 (Tyr-705), phospho-STAT3 (Ser-723), phospho-STAT5 (Tyr-694), phospho-JAK1 (Tyr-1034), phospho-JAK2 (Tyr-1007), phospho-EGFR (Tyr-1068), phospho-Src (Tyr-418), STAT1, STAT3, STAT5, JAK1, JAK2, EGFR, Src, Bcl-2, cyclin D1, c-Myc, survivin, MMP2, and MMP9 antibodies were from Signalway Antibody (Baltimore, MD).

Techniques: Modification, Recombinant, Incubation, Purification, Western Blot, Phospho-proteomics, In Vitro, Immunoprecipitation, Mutagenesis, Transfection, Plasmid Preparation, Binding Assay

Journal: Pharmacology Research & Perspectives

Article Title: 2‐Deoxy‐ d ‐glucose induces deglycosylation of proinflammatory cytokine receptors and strongly reduces immunological responses in mouse models of inflammation

doi: 10.1002/prp2.940

Figure Lengend Snippet: 2‐Deoxy‐D‐glucose (2‐DG) inhibits interleukin (IL)‐6 signalling and N ‐linked glycosylation of its receptor gp130. (A) Wildtype mouse embryonic fibroblasts (MEFs) were incubated in complete medium, glucose‐depleted medium (Glc‐), or medium with 25 mM 2‐DG for 5 h and then stimulated with IL‐6 and/or soluble IL‐6 receptor (sIL‐6R) (0.6 µg/mL each) for the indicated periods. Activating phosphorylation of Janus kinases (JAK1, JAK2, and TYK2) and expression of gp130 were assessed by immunoblotting. (B) Wildtype MEFs were incubated in medium with 25 mM 2‐DG or glucose‐depleted medium for the indicated periods. Band shifts of gp130 were assessed by immunoblotting. Glycopeptidase F (GP‐F) extract was used as a control for inhibition of N ‐linked glycosylation. (C) Wildtype MEFs were incubated with or without 25 mM 2‐DG for 5 h and whole cell extracts were precipitated with concanavalin A (ConA) agarose. ConA‐bound proteins were subjected to immunoblotting with an anti‐gp130 antibody. WCE, control whole cell extracts subjected to immunoblot analysis. (D) Wildtype MEFs were incubated with or without 25 mM 2‐DG for 5 h and then whole cell extracts were immunoprecipitated with the anti‐gp130 antibody and subjected to ConA staining. (E) Wildtype MEFs were incubated with or without 25 mM 2‐DG for 5 h and whole cell extracts were subjected to ConA and wheat germ agglutinin (WGA) staining. (F) Wildtype MEFs were incubated in medium with 25 mM 2‐DG or 100 µg/mL cycloheximide for the indicated periods. Expression of gp130 was assessed by immunoblotting. (G) Binding of IL‐6 to its receptor in the presence or absence of 2‐DG was assessed using FITC‐conjugated IL‐6 by flow cytometry analysis of human acute monocytic leukemia cells (THP1, left panel). The same cells were subjected to immunoblotting using the anti‐gp130 antibody as described in (B)

Article Snippet: Antibodies against gp130 (1:1000; Merck Life Science), phospho‐JAK1 (1:1000, Tyr 1022/1023/p‐JAK1; Cell Signaling Technology, Danvers, MA, USA), JAK1 (1:1000; Merck Life Science), phospho‐JAK2 (1:1000; Tyr 1007/1008/p‐JAK2; Cell Signaling Technology), JAK2 (1:1000; Merck Life Science), phospho‐TYK2 (1:1000; Tyr1054/1055/pTyk2; Cell Signaling Technology), TYK2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), phospho‐STAT3 (1:1000; Tyr 705/p‐STAT3; Cell Signaling Technology), STAT3 (1:1000; Cell Signaling Technology), phospho‐STAT1 (1:1000; Tyr 701/p‐STAT1; Cell Signaling Technology), STAT1 (1:1000; Santa Cruz Biotechnology), TNFR1 (1:1000; Cell Signaling Technology), IFNGR‐α (1:1000; Santa Cruz Biotechnology), α‐tubulin (1:1000; Sigma‐Aldrich, St. Louis, MO, USA), and β‐actin (1:1000; Sigma‐Aldrich) were used for immunoblot analyses.

Techniques: Incubation, Expressing, Western Blot, Inhibition, Immunoprecipitation, Staining, Binding Assay, Flow Cytometry

Journal: Pharmacology Research & Perspectives

Article Title: 2‐Deoxy‐ d ‐glucose induces deglycosylation of proinflammatory cytokine receptors and strongly reduces immunological responses in mouse models of inflammation

doi: 10.1002/prp2.940

Figure Lengend Snippet: 2‐Deoxy‐D‐glucose (2‐DG) inhibits the functions of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β and interferon (IFN)‐γ. (A) HeLa cells were transfected with Stat3 Luc or NF‐κB Luc reporter plasmids or phRL‐TK (internal control). After 24 h, cells were treated with or without 25 mM 2‐DG for 8 h and then with 0.4 µg/mL IL‐6 with soluble IL‐6 receptor (sIL‐6R) (Stat3 Luc), 50 ng/mL IL‐1β (NF‐κB Luc), or 100 ng/mL TNF‐α (NF‐κB Luc) for 4 h. After stimulation, relative luciferase activity was evaluated. Results were analysed using one‐way ANOVA followed by Tukey's post hoc test. **** p < .0001, *** p < .001, ** p < .01 and * p < .05. n.s.; not significant. Graphs are presented as the mean ±s.d. (n = 3). (B) HeLa cells were incubated in medium with 25 mM 2‐DG for the indicated periods. The band shift of TNF receptor 1 (TNFR1) was determined by immunoblotting. Glycopeptidase F (GP‐F) extract was used as a control for inhibition of N ‐linked glycosylation. (C) Binding of TNF‐α to its receptor in the presence or absence of 2‐DG was determined using FITC‐conjugated TNF‐α by flow cytometric analysis of THP1 cells (left panel). The same cells were subjected to immunoblotting using an anti‐TNFR1 antibody as described in (B). (D) Wildtype mouse embryonic fibroblasts (MEFs) were stimulated with 3000 U/mL IFN‐γ for the indicated times. Activating phosphorylation of Janus kinases (JAK1, JAK2, and TYK2) was assessed by immunoblotting. (E) HeLa cells were incubated in medium with 25 mM 2‐DG for the indicated periods. Band shift of IFN‐γ receptor α‐chain (IFNGRα) was assessed by immunoblotting. Tunicamycin and GP‐F extracts were used as controls for inhibition of N ‐linked glycosylation

Article Snippet: Antibodies against gp130 (1:1000; Merck Life Science), phospho‐JAK1 (1:1000, Tyr 1022/1023/p‐JAK1; Cell Signaling Technology, Danvers, MA, USA), JAK1 (1:1000; Merck Life Science), phospho‐JAK2 (1:1000; Tyr 1007/1008/p‐JAK2; Cell Signaling Technology), JAK2 (1:1000; Merck Life Science), phospho‐TYK2 (1:1000; Tyr1054/1055/pTyk2; Cell Signaling Technology), TYK2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), phospho‐STAT3 (1:1000; Tyr 705/p‐STAT3; Cell Signaling Technology), STAT3 (1:1000; Cell Signaling Technology), phospho‐STAT1 (1:1000; Tyr 701/p‐STAT1; Cell Signaling Technology), STAT1 (1:1000; Santa Cruz Biotechnology), TNFR1 (1:1000; Cell Signaling Technology), IFNGR‐α (1:1000; Santa Cruz Biotechnology), α‐tubulin (1:1000; Sigma‐Aldrich, St. Louis, MO, USA), and β‐actin (1:1000; Sigma‐Aldrich) were used for immunoblot analyses.

Techniques: Transfection, Luciferase, Activity Assay, Incubation, Electrophoretic Mobility Shift Assay, Western Blot, Inhibition, Binding Assay