Journal: bioRxiv

Article Title: Glia multitask to compensate for neighboring glial cell dysfunction

doi: 10.1101/2024.09.06.611719

Figure Lengend Snippet: ( A-B ) Control VNCs with CG (green), astrocytes (magenta), and Crz neurons (anti-Crz, gray) show normal Crz+ neuronal cell bodies and projections at L3 (A), but by 6 hours after pupal formation (h APF) the cell bodies are mostly cleared by CG and the projections are cleared by astrocytes (B). ( C-D ) When CG expressing RNAi against Spz3 exhibit disrupted CG morphology, more Crz+ neuron cell bodies remain, while the projections are still cleared. Scale bars = 10 µm. White arrowheads denote Crz+ neuronal projections while the yellow arrowheads denote the Crz+ neuronal cell bodies. ( E ) Quantification of the number of Crz+ neuronal cell bodies at L3 and 6h APF and ( F ) Crz neurite debris remaining at 6h APF. ( G-H ) Control brains with CG (green), EG (magenta), neuronal nuclei (blue), and or88a-CD2 (anti-ratCD2, gray) show or88a+ neuronal projections in the antenna lobe in uninjured animals (G), but these projections are cleared by EG following injury partially at 3 days post injury (DPI) and fully by 5 DPI (H). ( I-J ) When CG morphology is disrupted with Spz3 RNAi, or88a+ neurons are still cleared by EG. Scale bars = 10 µm. ( K-L ) Quantification of the or88a+ volume normalized to the volume of the antennal lobe in uninjured control and Spz3 knockdown animals (K) and at 3 and 5 DPI (L). ( M ) Control brains with CG (green) exposed to 70 kDa Dextran (magenta) successfully block Dextran from the L3 CNS. ( N ) When CG express Spz3 RNAi , Dextran is still excluded from the L3 CNS, suggesting an intact BBB and functioning SPG. ( O ) Quantification of the area of Dextran within the L3 CNS. N = numbers of animals, denoted in each corresponding bar. (n.s.) Not significant. (*) P < 0.5, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. (E, F, K) Unpaired t-test, (L,O) One-Way ANOVA with multiple comparisons.

Article Snippet: The following primary antibodies were used: chicken anti-GFP (1:1000; Antibodies Inc), rat anti- mCherry (1:1000, Invitrogen); rabbit anti-DsRed (1:1000; Clontech) rat anti-Elav (1:100; Developmental Studies Hybridoma Bank [DSHB], 7E8A10), mouse anti-Elav (1:100, DSHB, 9F8A9), Anti-β-Galactosidase (1:1000; Promega, Z3781), mouse anti-Repo (1:5; DSHB, 8D12), mouse anti-Prospero (1:100; DSHB, MR1A), rabbit anti-DCP1 (1:100; Cell Signaling, 9578S), rabbit anti-corazonin (1:500, Freeman Lab), mouse anti-nc82 (1:20; DSHB, nc82), mouse anti- ratCD2 (1:200; BioRad, MCA154GA), rabbit anti-p35 (1:1000, Fisher Scientific, NB10056153T).

Techniques: Control, Expressing, Knockdown, Blocking Assay

Journal: bioRxiv

Article Title: The African Swine Fever Virus gene MGF_360-4L inhibits interferon signaling by recruiting mitochondrial selective autophagy receptor SQSTM1 degrading MDA5 antagonizing innate immune responses

doi: 10.1101/2024.09.09.612163

Figure Lengend Snippet: MGF_360-4L promotes the autophagic degradation of MDA5. (A) HEK293T cells were transfected with increasing doses of pHA-MGF_360-4L and pFlag-MDA5. The cells were then collected and subjected to Western blotting with the indicated antibodies to analyze the effect of MGF_360-4L on MDA5 expression. (B-C). HEK293T cells were transfected with increasing doses of pHA-MGF_360-4L, and the transcript levels of MGF_360-4L (B) and MDA5 (C) were measured via RT‒qPCR. (D) HEK293T cells were cotransfected with pFlag-MDA5 and with or without pHA-MGF_360-4L, followed by treatment with DMSO (negative control), NH 4 Cl (20 mM), MG-132 (10 μM), 3-MA (10 mM), or Z-VAD-FMK (20 μM) for 24 h. Then, the cells were collected, and the protein expression levels of MDA5 and MGF_360-4L were analyzed via Western blotting. (E) HEK293T cells were transfected with pHA-MGF_360-4L or pHA-VEC and infected with VSV at the indicated time points. Cell samples were collected, and the protein expression levels of LC3 and P62 were assessed via Western blotting. (F) HeLa cells were cotransfected with Mcheey-LC3B and EGFP-LC3B with or without pHA-MGF_360-4L. The colocalization of pHA-MGF_360-4L and Mcheey-LC3B was observed via fluorescence microscopy. Scale bars, 10 μm. Fluorescence intensity profiles were measured along the drawn measurement line. (G) HEK293T cells were transfected with increasing doses of pHA-MGF_360-4L, followed by treatment with or without the autophagy inhibitor CQ (50 μM). Protein expression was assessed by Western blotting.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (USA): rabbit anti-DYKDDDK-Tag (CST, 14793S), mouse anti-DYKDDDK-Tag (CST, 8146), rabbit anti-HA (CST, 3724), mouse anti-HA (CST, 2367), rabbit anti-His (CST, 12698), rabbit anti-Myc (CST, 13987), and mouse anti-Myc (CST, 2276); rabbit anti-Rig-I (CST, 3743), rabbit anti-MDA5 (CST, 5321), rabbit anti-MAVS (CST, 24930), rabbit anti-TRAF3 (CST, 33640), rabbit anti-IKKε (CST, 96794), rabbit anti-p-IKKε (CST, 8766), rabbit anti-TBK1 (CST, 3504), rabbit anti-p-TBK1 (CST, 5483), rabbit anti-IRF3 (CST, 4302), rabbit anti-p-IRF3 (CST, 29047), rabbit anti-IRF7 (CST, 72073), rabbit anti-p-IRF7 (CST, 24129), rabbit anti-LC3A/B (CST, 12741), rabbit anti-LC3B (CST, 2775), rabbit anti-SQSTM1/p62 (CST, 23214), rabbit anti-NDP52 (CST, 60732), rabbit anti-NIX (CST, 12396), mouse anti-PARKIN (CST, 4211), rabbit anti-PINK1 (CST, 6946), rabbit anti-BNIP3 (CST, 44060), rabbit anti-ATG5 (CST, 12994), rabbit anti-ATG7 (CST, 8558), rabbit anti-ATG12 (CST, 4180), and rabbit anti-ATG16L1 (CST, 8089) antibodies.

Techniques: Transfection, Western Blot, Expressing, Negative Control, Infection, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: The African Swine Fever Virus gene MGF_360-4L inhibits interferon signaling by recruiting mitochondrial selective autophagy receptor SQSTM1 degrading MDA5 antagonizing innate immune responses

doi: 10.1101/2024.09.09.612163

Figure Lengend Snippet: MGF_360-4L recruits the selective autophagy receptor p62 to induce mitophagy. (A-B) HEK293T cells were cotransfected with pHA-MGF_360-4L and Myc-OPTN, Myc-NBR1, Myc-TOLLIP, Myc-NDP52, or Myc-p62, and the interactions between proteins were detected by Co-IP. (C) GST-tagged MGF_360-4L protein (10 μg) or GST antigen was added to GST-Sepharose 4B beads, which were incubated for 3 h at room temperature, followed by binding with His-tagged p62 protein (10 μg). The samples were subjected to Western blotting analysis to detect direct interactions between MGF_360-4L-GST and p62-His. (D-E) HEK293T cells were transfected with pFlag-MDA5 with or without pHA-MGF_360-4L, followed by the addition of siRNA targeting p62 or ATG16L1 or a scramble siRNA as a control. The cells were collected, and the protein expression levels were analyzed via Western blotting. (F-H) HeLa cells were transfected with pHA-MGF_360-4L and pMyc-p62, NDP52 or empty vector. The colocalization of MGF_360-4L with p62 or NDP52 was detected via confocal microscopy. Fluorescence intensity profiles were measured along the drawn measurement line.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (USA): rabbit anti-DYKDDDK-Tag (CST, 14793S), mouse anti-DYKDDDK-Tag (CST, 8146), rabbit anti-HA (CST, 3724), mouse anti-HA (CST, 2367), rabbit anti-His (CST, 12698), rabbit anti-Myc (CST, 13987), and mouse anti-Myc (CST, 2276); rabbit anti-Rig-I (CST, 3743), rabbit anti-MDA5 (CST, 5321), rabbit anti-MAVS (CST, 24930), rabbit anti-TRAF3 (CST, 33640), rabbit anti-IKKε (CST, 96794), rabbit anti-p-IKKε (CST, 8766), rabbit anti-TBK1 (CST, 3504), rabbit anti-p-TBK1 (CST, 5483), rabbit anti-IRF3 (CST, 4302), rabbit anti-p-IRF3 (CST, 29047), rabbit anti-IRF7 (CST, 72073), rabbit anti-p-IRF7 (CST, 24129), rabbit anti-LC3A/B (CST, 12741), rabbit anti-LC3B (CST, 2775), rabbit anti-SQSTM1/p62 (CST, 23214), rabbit anti-NDP52 (CST, 60732), rabbit anti-NIX (CST, 12396), mouse anti-PARKIN (CST, 4211), rabbit anti-PINK1 (CST, 6946), rabbit anti-BNIP3 (CST, 44060), rabbit anti-ATG5 (CST, 12994), rabbit anti-ATG7 (CST, 8558), rabbit anti-ATG12 (CST, 4180), and rabbit anti-ATG16L1 (CST, 8089) antibodies.

Techniques: Co-Immunoprecipitation Assay, Incubation, Binding Assay, Western Blot, Transfection, Control, Expressing, Plasmid Preparation, Confocal Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Active EB1 surges promote tubulin influx into the growing outer segments of the bipartite olfactory cilia in Drosophila

doi: 10.1101/2024.09.10.612170

Figure Lengend Snippet: A) Copurification of EB1:GFP from the chaGal4>EB1:GFP expressing Drosophila head extracts with the recombinant Glutathione S-transferase (GST)-tagged, KLP68D tail (GST-KLP68DT) fragment using affinity chromatography. The arrow indicates the EB1:GFP band. B) The immune-coprecipitation (IP) of the recombinant KLP68D from the head extracts of chaGal4>Klp68D:YFP and chaGal4>Klp68D(ΔT)YFP, expressing UAS-EB1FLAG using anti-FLAG. The arrow indicates a full-length KLP68DYFP band.

Article Snippet: Proteins from these gels were transferred onto a previously activated PVDF membrane (Hybond-P, GE Healthcare Ltd) in an electro-blotting apparatus (Bio-Rad, USA) following the supplier’s protocol and incubated in different primary antisera solutions as the following: anti-GFP Rabbit (dilution, 1:500; #3999 Bio Vision Inc., CA, USA) or anti-FLAG Rabbit (1:500; #F7425 Sigma-Aldrich, USA) Or anti-GST mouse (1:1000, Bioklone Biotech Pvt.

Techniques: Copurification, Expressing, Recombinant, Affinity Chromatography