Journal: Cells

Article Title: Salidroside Derivative SHPL-49 Exerts Anti-Neuroinflammatory Effects by Modulating Excessive Autophagy in Microglia

doi: 10.3390/cells14060425

Figure Lengend Snippet: SHPL-49 exerts anti-inflammatory effects by suppressing the NF-κB signaling pathway. ( A ) Immunohistochemical staining images of NF-κB and IL-6 in brain tissue sections from rats subjected to pMCAO and treated with SHPL-49 (15 mg/kg) and ED (7.5 mg/kg) for three days, Scale = 100 μm. ( B ) The IODs for NF-κB and IL-6 immunohistochemistry in brain tissue sections for each treatment group. ( C ) Representative Western blot of NF-κB in the nucleus and cytoplasm of OGD-BV2 cells after treatment with SHPL-49 (200 μM). ( D ) Quantification of the Western blot results. ( E ) Immunofluorescence staining pattern of NF-κB in OGD-BV2 cells following treatment with SHPL-49 (200 μM); Scale = 10 μm. ( F ) Quantification of the average fluorescence intensity of NF-κB in the nucleus of BV2 cells. ( G ) Representative Western blot of p-IKKβ, IKKβ, p-IκBα, and IκBα proteins in OGD-BV2 cells after treatment with SHPL-49 (200 μM). ( H ) Quantification of the Western blot results. ( I ) The protein expression levels of inflammatory factors IL-6, IL-1β, and iNOS in the supernatant of OGD-BV2 cells following treatment with SHPL-49 (200 μM). Data are presented as means ± SD with n = 6 per group. *** p < 0.001 vs. Ctrl; ## p < 0.01, ### p < 0.001 vs. OGD.

Article Snippet: Membranes were incubated with 5% nonfat milk (Beyotime, Shanghai, China, P0216) or BSA (Beyotime, Shanghai, China, ST023) followed by overnight incubation at 4 °C with the following primary antibodies: NF-κB (1:1000, Cell Signaling Technology, MA, USA, D14E12), p-IKKβ (1:1000, UpingBio, Hangzhou, Zhejiang, China, YP-Ab-14443), IKKβ (1:1000, Boster, Wuhan, China, BM4875), p-IκBα (1:1000, UpingBio, Zhejiang, China, YP-Ab-01253), IKBα (1:1000, UpingBio, Zhejiang, China, YP-Ab-01830), P62/SQSTM1 (1:1000, Boster, Wuhan, China, BM4385), LC3B (1:200, PTMab, Zhejiang, China, PTM-6384), ATG5 (1:1000, Cell Signaling Technology, MA, USA, D5F5U), Bclin1 (1:1000, Cell Signaling Technology, MA, USA, D40C5), LAMP2 (1:200, UpingBio, Zhejiang, China, YP-Ab-14094), Bax (1:1000, Cell Signaling Technology, MA, USA, 14796), Bcl-2 (1:1000, AiFang biologic, Hunan, China, AF0060), Cleaved caspase-3 (1:1000, Cell Signaling Technology, MA, USA, 9661S), and Cleaved caspase-9 (1:1000, Cell Signaling Technology, MA, USA, 9507S).

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Western Blot, Immunofluorescence, Fluorescence, Expressing

Journal: Antioxidants

Article Title: Gastro-Protective Effects of Albizia anthelmintica Leaf Extract on Indomethacin-Induced Gastric Ulcer in Wistar Rats: In Silico and In Vivo Studies

doi: 10.3390/antiox10020176

Figure Lengend Snippet: Effects of indomethacin alone and with oral pre-treatments of famotidine or A. anthelmintica (200, 100 mg/kg) on ( A ) gastric inhibitor of nuclear factor kappa-B kinase subunit beta (IKKB), ( B ) gastric nuclear factor KB (NF-κB), ( C ) gastric TNF-α, and ( D ) gastric IL-6 in rats. Statistical analyses were carried out by one-way ANOVA followed by Tukey’s post hoc test, mean ± SEM. * Significantly different from control group at p < 0.05. @ Significantly different from indomethacin alone group at p < 0.05.

Article Snippet: Primary rabbit polyclonal antibodies against inhibitor of nuclear factor kappa-B kinase subunit beta (IKK-β, catalogue number: PA2036-1) and rabbit monoclonal antibodies against β-actin (catalogue number: M01263) were purchased from Boster Biological Technology (Pleasanton, CA, USA) and diluted at 1:1000 and 1:5000, respectively.

Techniques: Control

Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor–mediated phagosome formation and maturation in macrophages

doi: 10.1091/mbc.E17-08-0523

Figure Lengend Snippet: Structural alteration of phagosomal SNAP-23 caused by IKK2 reduced the efficiency of phagosome maturation. (A) J774 cells were transiently cotransfected with a SNAP-23 tG-S1-tR-S2 probe and Myc-IKK2, which was followed by determination of the FRET efficiency on the plasma membrane of living cells. (B, C) J774 cells cotransfected with SNAP-23 probes and Myc-IKK2 were incubated with IgG-opsonized zymosan particles at 37°C for 20 min, in the presence (C) or absence (B) of SC-514 at the indicated concentrations, and the FRET efficiency on the phagosome membrane of living cells was then measured. Data presented are the means ± SEs of four to five independent experiments. (D) J774 cells labeled with RB–dextran were transiently transfected with an empty Myc vector, Myc-IKK2, or Myc-IKK2-KD (kinase dead) and then subjected to phagosome-lysosome fusion assays as described in . Data presented are the means ± SEs of six independent experiments. Statistical analyses were carried out using one-way ANOVA with Tukey’s post-hoc test (for experiments with more than two groups, panels B, C, and D).

Article Snippet: The remaining antibodies were obtained from commercial sources as follows: anti-syntaxin 13 from Stressgen (Victoria, Canada); anti-syntaxin 3, anti-syntaxin 4, and anti-SNAP-23 from Sigma-Aldrich (St. Louis, MO); anti-VAMP8 from Synaptic Systems (Göttingen, Germany); anti-IKK2 (clone 42D1) from Abnova (Taipei, Taiwan); and anti-CD64 (N-19) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Membrane, Incubation, Labeling, Transfection, Plasmid Preparation

Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor–mediated phagosome formation and maturation in macrophages

doi: 10.1091/mbc.E17-08-0523

Figure Lengend Snippet: Structural alteration of phagosomal SNAP-23 in IFN-γ–activated macrophages was partially dependent on IKK2 activity and caused a delay in phagosome maturation. (A, B) J774 cells were transiently transfected with SNAP-23 probes and incubated in the presence or absence of IFN-γ (100 U/ml). Eighteen hours after transfection, the FRET efficiency of living cells on the plasma membrane (A) or on the membrane of phagosomes containing IgG-opsonized zymosan particles (B) was analyzed as described under Materials and Methods . Data presented are the means ± SEs of 7 to 10 independent experiments. (C) J774 cells coexpressing the SNAP-23 tG-S1-tR-S2 probe and a Myc-tagged construct (empty vector or Myc-IKK2) were incubated in the presence or absence of IFN-γ (100 U/ml). Eighteen hours after transfection, the cells were incubated with IgG-opsonized zymosan particles in the presence or absence of SC-514 (30 μM) at 37°C for 20 min, and, subsequently, the FRET efficiency on the phagosome membranes of living cells was analyzed. Data presented are the means ± SEs of three independent experiments. (D) J774 cells labeled with RB–dextran were incubated in the presence or absence of IFN-γ (100 U/ml) for 18 h and then subjected to phagosome-lysosome fusion assays as described in . IFN-γ–activated cells were preincubated in the presence or absence of SC-514 (30 μM) at 37°C for 20 min before and during the assay. Data presented are the means ± SEs of four independent experiments. Statistical analyses were carried out using two-tailed, paired Student’s t tests (for experiments with two groups, panel A) or one-way ANOVA with Tukey’s post-hoc test (for experiments with more than two groups, panels B, C, and D).

Article Snippet: The remaining antibodies were obtained from commercial sources as follows: anti-syntaxin 13 from Stressgen (Victoria, Canada); anti-syntaxin 3, anti-syntaxin 4, and anti-SNAP-23 from Sigma-Aldrich (St. Louis, MO); anti-VAMP8 from Synaptic Systems (Göttingen, Germany); anti-IKK2 (clone 42D1) from Abnova (Taipei, Taiwan); and anti-CD64 (N-19) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay, Transfection, Incubation, Membrane, Construct, Plasmid Preparation, Labeling, Two Tailed Test

Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor–mediated phagosome formation and maturation in macrophages

doi: 10.1091/mbc.E17-08-0523

Figure Lengend Snippet: Schematic representation of SNAP-23–mediated regulation of phagosome formation and maturation during FcR-mediated phagocytosis in macrophages. Formation of a phagosome containing an IgG-opsonized particle is induced by SNAP-23 without phosphorylation at Ser95. Phosphorylation of SNAP-23 by other factors, such as PKC, suppresses its ability to regulate phagosome formation. In the presence of both IgG-opsonized and nonopsonized particles, a non-FcR–mediated phagocytosis pathway may allow phosphorylated SNAP-23 to induce phagosome formation, resulting in the suppression of FcR-mediated phagocytosis. Although SNAP-23 in resting macrophages facilitates phagosome maturation, this process is suppressed or delayed by the phosphorylation of SNAP-23 at Ser95 by IKK2 in IFN-γ–activated macrophages (see Discussion ).

Article Snippet: The remaining antibodies were obtained from commercial sources as follows: anti-syntaxin 13 from Stressgen (Victoria, Canada); anti-syntaxin 3, anti-syntaxin 4, and anti-SNAP-23 from Sigma-Aldrich (St. Louis, MO); anti-VAMP8 from Synaptic Systems (Göttingen, Germany); anti-IKK2 (clone 42D1) from Abnova (Taipei, Taiwan); and anti-CD64 (N-19) from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: