Journal: Frontiers in Cell and Developmental Biology

Article Title: The BEACH Domain Is Critical for Blue Cheese Function in a Spatial and Epistatic Autophagy Hierarchy

doi: 10.3389/fcell.2019.00129

Figure Lengend Snippet: bchs’ neurodegenerative phenotype is sensitive to autophagy-modulating drugs and genetic enhancement of autophagy. (A–D) Larvae of the indicated genotypes were fed on standard food containing either (A) rapamycin, (B) Wortmannin, or (C) 3-methyladenine. All third instar larval motor neurons were labeled as shown in the example in (D) with anti-Futsch (22C10; red), and co-labeled with anti-GFP to detect eve>GFP in aCC and RP2 motor neurons (green; arrows). RP2 is absent in the bchs mutant (right panel). Scalebar = 40 μM. Percent survival of RP2 motor neurons over ∼100 hemisegments was calculated for each treatment, and experiments were performed in triplicate. Chi-square test was used to determine statistical significance; * p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (E) Over-expression of atg7 shown by RT-PCR on total RNA from adult heads of wild type (WT, CantonS), atg7[d77]/+ heterozygotes, and elav-Gal4>atg7, using primers specific for atg7 mRNA and rp49 rRNA as loading control. (F) Over-expression of atg7 increases RP2 motor neuron survival in bchs17(M) and bchs58(M) (lanes 3 vs. 2 and 6 vs. 5). A single allelic deletion of atg7 by itself (lane 9) gives a similar rate of RP2 death as bchs58(M) and mildly exacerbates it (lanes 5 vs. 7) but does not significantly change bchs17(M) (lanes 2 vs. 4). Experiments were performed in triplicate, and N > 100 (hemisegments) for each genotype. All genotypes include the driver/reporter combination eve>GFP in homozygosity. Error bars represent standard deviation, and Chi-square test was used to calculate statistical significance. * p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Embryos were collected every 3 h on apple juice agar plates, and incubated at 25°C for 24 h; hatching first instar larvae were transferred into 2.0 mL Eppendorf tubes with food containing 0.05% (v/v) ethanol (vehicle control), 1 μM rapamycin (AG Scientific), 0.2 or 2 μM Wortmannin (AG Scientific; R-1018) or 5 mM 3-methyladenine (Calbioche ® m, Merck Millipore, 189490), and dissected as third instar larvae.

Techniques: Labeling, Mutagenesis, Over Expression, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: The BEACH Domain Is Critical for Blue Cheese Function in a Spatial and Epistatic Autophagy Hierarchy

doi: 10.3389/fcell.2019.00129

Figure Lengend Snippet: Ubiquitinated aggregates accumulate in neuronal termini of bchs larval neuromuscular junctions and can be reduced by rapamycin feeding. (A) Third instar larvae were dissected and immunostained for anti-HRP (a pan-neuronal marker) and anti-poly-ubiquitin. Scale-bar = 20 μm. (B) Percentages of neuromuscular junctions with ubiquitinated inclusions in each of three size groups (see Materials and Methods) were calculated for the different genotypes. Experiments were performed in duplicate, and error bars represent S.E.M. Chi-square test was used to determine pairwise statistical significance between frequency of occurrence of a given aggregate class in bchs vs. the same class in the wild-type control. * p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Larvae of the indicated genotypes were fed on (C) rapamycin, (D) Wortmannin, or (E) 3-methyladenine (see Materials and Methods) before repeating the same procedures as in (B) for analysis. Stars in (A) show significance of differences between classes in each of the genotypes and wild-type (+/+). Stars in (C) and (E) show significance for pairwise comparisons of numbers of ubiquitinated inclusions in the same genotype in (B) without drug.

Article Snippet: Embryos were collected every 3 h on apple juice agar plates, and incubated at 25°C for 24 h; hatching first instar larvae were transferred into 2.0 mL Eppendorf tubes with food containing 0.05% (v/v) ethanol (vehicle control), 1 μM rapamycin (AG Scientific), 0.2 or 2 μM Wortmannin (AG Scientific; R-1018) or 5 mM 3-methyladenine (Calbioche ® m, Merck Millipore, 189490), and dissected as third instar larvae.

Techniques: Marker

Journal: Veterinary research

Article Title: Chicken interferon-induced transmembrane proteins inhibit Newcastle disease virus infection by affecting viral entry and W protein expression.

doi: 10.1186/s13567-025-01530-y

Figure Lengend Snippet: Figure 7 chIFITMs reduce NDV W protein expression. A NDV W protein expression was inhibited in chIFITMs by using a laboratory-prepared W polyclonal antibody and HA/Histone H3 primary antibodies with HRP-conjugated goat anti-rabbit/mouse IgG (H + L). B Schematic of pcDNA3.1-W recombinant plasmids and western blot confirmation of W protein expression. C Co-transfection of chIFITMS with the NDV W recombinant expression plasmid. Impact of chIFITM overexpression on W protein levels, as determined by His/HA/α-tubulin primary antibodies (W-His, chIFITM-HA) and HRP-conjugated goat anti-rabbit/mouse IgG (H + L). D Western blot analysis of the effects of the human proteasome inhibitor MG132 (5 μM) and the autophagy inhibitor 3-MA (5 mM) on chIFITM-mediated W protein inhibition. E Co-IP study of the interaction between chIFITMs and W protein.

Article Snippet: 3-MA (189490-50 MG) and MG132 (474790-1 MG) were purchased from MCE.

Techniques: Expressing, Recombinant, Western Blot, Cotransfection, Plasmid Preparation, Over Expression, Inhibition, Co-Immunoprecipitation Assay