Journal: Autophagy

Article Title: Zika virus NS2A protein induces the degradation of KPNA2 (karyopherin subunit alpha 2) via chaperone-mediated autophagy

doi: 10.1080/15548627.2020.1823122

Figure Lengend Snippet: List of primers used in this study

Article Snippet: The primary mouse monoclonal antibodies against KPNA1 (Santa Cruz Biotechnology, sc-101,292), KPNA2 (Santa Cruz Biotechnology, sc-55,537), hemagglutinin (HA) tag (ThermoFisher Scientific, 26,183), GFP (Biolegend, 75,818–584), HSPA8/HSC70 (Santa Cruz Biotechnology, sc-7298), ubiquitin (Santa Cruz Biotechnology, sc-8017), GAPDH (Santa Cruz Biotechnology, sc-365,062), and TUBB1/β-tubulin (Sigma, T7816), and rabbit polyclonal antibodies against ZIKV NS4B (GeneTex, GTX133311), ZIKV E (GeneTex, GTX133314), NS2B (GeneTex, GTX133318), NS5 (GeneTex, GTX133329) and LAMP2A (Boster, M01573) were used in this study.

Techniques:

Journal: Cell Reports Medicine

Article Title: Targeting phenylpyruvate restrains excessive NLRP3 inflammasome activation and pathological inflammation in diabetic wound healing

doi: 10.1016/j.xcrm.2023.101129

Figure Lengend Snippet: Phenylpyruvate upregulated NLRP3 palmitoylation by binding to the PPT1 protein (A) Schematic view of NLRP3 (full length) and the location of the S-palmitoylation sites. (B) ABE assay and immunoblot analysis were used to evaluate NLRP3 palmitoylation in BMDMs treated with or without palmitic acid (n = 3). (C) Immunoblot and statistical analysis showing NLRP3 expression in BMDMs treated with 2BP at the indicated concentrations (0, 50, 100, and 150 μM) for 24 h (n = 3). (D) Immunoblot and statistical analysis showing NLRP3 expression in BMDMs treated with 2BP (100 μM) for 0, 8, 16, and 24 h (n = 3). (E) Immunostaining of the location of LAMP2 (indicating lysosomes), PPT1, and NLRP3 in macrophages. The nuclei were stained with Hoechst dye. Scale bar, 10 μm. (F and G) Immunoprecipitation (IP) and immunoblot analysis were used to detect the endogenous NLRP3 and PPT1 association in macrophages (n = 3). (H) ABE assay and immunoblot analysis were used to evaluate NLRP3 palmitoylation in BMDMs with Ppt1 knockdown (n = 3). (I) Mouse embryo fibroblasts (MEFs) were transfected with WT NLRP3 or the indicated NLRP3 mutants for 24 h with or without knockdown of Ppt1. ABE assay and immunoblot analysis showing palmitoylation levels of the indicated NLRP3 mutants (n = 3). (J) ABE assay and immunoblot analysis were used to determine NLRP3 palmitoylation levels in BMDMs treated with increasing phenylpyruvate concentrations (n = 3). (K) Determination of NLRP3 palmitoylation levels in BMDMs treated with 400 μM phenylpyruvate and then transfected with the Ppt1 WT plasmid or the MUT plasmid for 24 h (n = 3). (L) BMDMs were treated with 400 μM phenylpyruvate with or without 100 μM 2BP and then transfected with or without the Ppt1 MUT plasmid. ABE assay and immunoblot analysis determining NLRP3 palmitoylation levels in the indicated cells (n = 3). Data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01; n.s., not significant.

Article Snippet: Rat anti-mouse LAMP2 , Proteintech , Cat# 65052-1-Ig; RRID: AB_2881468.

Techniques: Binding Assay, Western Blot, Expressing, Immunostaining, Staining, Immunoprecipitation, Knockdown, Transfection, Plasmid Preparation

Journal: Cell Reports Medicine

Article Title: Targeting phenylpyruvate restrains excessive NLRP3 inflammasome activation and pathological inflammation in diabetic wound healing

doi: 10.1016/j.xcrm.2023.101129

Figure Lengend Snippet:

Article Snippet: Rat anti-mouse LAMP2 , Proteintech , Cat# 65052-1-Ig; RRID: AB_2881468.

Techniques: Recombinant, Transfection, Lysis, cDNA Synthesis, Real-time Polymerase Chain Reaction, BIA-KA, Enzyme-linked Immunosorbent Assay, Software

Journal: Bioengineering

Article Title: Strontium Ranelate Inhibits Osteoclastogenesis through NF-κB-Pathway-Dependent Autophagy

doi: 10.3390/bioengineering10030365

Figure Lengend Snippet: Strontium ranelate suppressed osteoclast differentiation through autophagy in vitro. ( a ) TRAP staining images after pre-osteoclasts received different treatments for 5 days. ( b ) Monodansylcadaverine staining images after pre-osteoclasts received different treatments for 5 days. ( c ) Representative images of autophagosomes captured with a transmission electron microscope after pre-osteoclasts received different treatments for 5 days. Western blot assay and relative protein expression of the osteoclast markers TRAF6, c-Fos, MMP-9, MMP-14, and CTSK ( d ) and the autophagic proteins Beclin1, ATG5, LAMP2, LC3-I, LC3-II, and p62 ( e ) after pre-osteoclasts received different treatments for 5 days. (n = 3, mean ± SD, ** p < 0.01, *** p < 0.001).

Article Snippet: After that, the slices were incubated with a primary antibody at 4 °C overnight, including RANK (1:100; Zen), osteoprotegerin (OPG; 1:200; Servicebio), TRAF6 (1:100; Servicebio), nuclear factor of activated T cells 2 (NFATc2; 1:500; Servicebio), c-Fos (1:400; Servicebio), MMP-14 (1:100; Zen), CTSK (1:1000; Servicebio), p-IKK α/β (1:100; Affinity), IκBα (1:100; Zen), p65 (1:200; Zen), Beclin1 (1:200; Boster), LC3 (1:500; CST), LAMP2 (1:100; Zen), ATG5 (1:50; Zen), and p62 (1:200; Boster).

Techniques: In Vitro, Staining, Transmission Assay, Microscopy, Western Blot, Expressing

Journal: Bioengineering

Article Title: Strontium Ranelate Inhibits Osteoclastogenesis through NF-κB-Pathway-Dependent Autophagy

doi: 10.3390/bioengineering10030365

Figure Lengend Snippet: Strontium ranelate inhibited osteoclast differentiation through NF-κB-pathway-dependent autophagy in vitro. ( a ) TRAP staining images after pre-osteoclasts received different treatments for 5 days. ( b ) Monodansylcadaverine staining images after pre-osteoclasts received different treatments for 5 days. ( c ) Representative images of autophagosomes captured with a transmission electron microscope after pre-osteoclasts received different treatments for 5 days. Western blot assay and relative protein expression of the osteoclast marker CTSK, the proteins central to the NF-κB pathway (p-IKKα/β, IκBα, and p65) ( d ), and the autophagic proteins Beclin-1, ATG5, LAMP2, LC3-I, LC3-II, and p62 ( e ) after pre-osteoclasts were treated with Bay 11-7082 for 5 days. ( f ) Western blot assay and relative protein expression of the proteins central to the NF-κB pathway after pre-osteoclasts were treated with SR for 5 days. (n = 3, mean ± SD, *** p < 0.001).

Article Snippet: After that, the slices were incubated with a primary antibody at 4 °C overnight, including RANK (1:100; Zen), osteoprotegerin (OPG; 1:200; Servicebio), TRAF6 (1:100; Servicebio), nuclear factor of activated T cells 2 (NFATc2; 1:500; Servicebio), c-Fos (1:400; Servicebio), MMP-14 (1:100; Zen), CTSK (1:1000; Servicebio), p-IKK α/β (1:100; Affinity), IκBα (1:100; Zen), p65 (1:200; Zen), Beclin1 (1:200; Boster), LC3 (1:500; CST), LAMP2 (1:100; Zen), ATG5 (1:50; Zen), and p62 (1:200; Boster).

Techniques: In Vitro, Staining, Transmission Assay, Microscopy, Western Blot, Expressing, Marker

Journal: Bioengineering

Article Title: Strontium Ranelate Inhibits Osteoclastogenesis through NF-κB-Pathway-Dependent Autophagy

doi: 10.3390/bioengineering10030365

Figure Lengend Snippet: Strontium ranelate regulated the expression of autophagic proteins in rats. ( a ) Immunohistochemical staining images of ATG5, Beclin1, LAMP2, LC3, and p62 at the pressure side of the first molars on day 7 in different groups. ( b ) The mean optical density (MOD) of ATG5, Beclin1, LAMP2, LC3, and p62 at the pressure side of the first molars on days 3, 7, and 14 in different groups. (n = 5, mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001). ( c ) Immunofluorescence staining images of ATG5, Beclin1, LAMP2, LC3, and p62 at the pressure side of the first molars on day 7 in different groups. The white dashed lines indicate the margin of the tooth root.

Article Snippet: After that, the slices were incubated with a primary antibody at 4 °C overnight, including RANK (1:100; Zen), osteoprotegerin (OPG; 1:200; Servicebio), TRAF6 (1:100; Servicebio), nuclear factor of activated T cells 2 (NFATc2; 1:500; Servicebio), c-Fos (1:400; Servicebio), MMP-14 (1:100; Zen), CTSK (1:1000; Servicebio), p-IKK α/β (1:100; Affinity), IκBα (1:100; Zen), p65 (1:200; Zen), Beclin1 (1:200; Boster), LC3 (1:500; CST), LAMP2 (1:100; Zen), ATG5 (1:50; Zen), and p62 (1:200; Boster).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Streptococcus lutetiensis Induces Autophagy via Oxidative Stress in Bovine Mammary Epithelial Cells

doi: 10.1155/2022/2549772

Figure Lengend Snippet: Antibody details.

Article Snippet: Anti-LAMP2 , AF1036 , Beyotime , Shanghai, China , 1 : 1000.

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Streptococcus lutetiensis Induces Autophagy via Oxidative Stress in Bovine Mammary Epithelial Cells

doi: 10.1155/2022/2549772

Figure Lengend Snippet: S. lutetiensis decreased pH in lysosomes to further block the autophagy flux. (a) To assess lysosomal pH, cells were stained with 100 nM LysoTracker Deep Red at 37°C for 30 min after being infected with S. lutetiensis . Scale bars: 20 μ m. (b) After being infected with S. lutetiensis , cells were stained with AO at 37°C to assess lysosomal pH. Scale bars: 20 μ m. (c–e) Protein levels of LAMP2, cathepsin D, and cathepsin L in MAC-T cells at various intervals after infection with S. lutetiensis . Upper panels: representative Western blot images; lower panels: quantitative analysis (mean ± SD, n = 3, ∗ represents the significance with the 0 h group, ∗ p < 0.05).

Article Snippet: Anti-LAMP2 , AF1036 , Beyotime , Shanghai, China , 1 : 1000.

Techniques: Blocking Assay, Staining, Infection, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Streptococcus lutetiensis Induces Autophagy via Oxidative Stress in Bovine Mammary Epithelial Cells

doi: 10.1155/2022/2549772

Figure Lengend Snippet: NAC efficiently decreased S. lutetiensis -induced autophagy in MAC-T. (a–f) Protein levels of LC3II/I, p62, Beclin1, LAMP2, CTSD, and CTSL. Upper panels: representative Western blot images; lower panels: quantitative analysis (mean ± SD, n = 3, ∗ represents the significance with the control group, # represents the significance with the treated group, ∗ p < 0.05, # p < 0.05; C: control; T: treatment; R: rapamycin; M: 3-MA; T+N: treatment + NAC). (g) Lysosome detection comparison between infected with S. lutetiensis with/without NAC. Scale bars: 20 μ m.

Article Snippet: Anti-LAMP2 , AF1036 , Beyotime , Shanghai, China , 1 : 1000.

Techniques: Western Blot, Infection