Journal: bioRxiv

Article Title: Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus

doi: 10.1101/2024.06.19.599730

Figure Lengend Snippet: (A) In silico prediction of signal peptide in Toll-9 protein sequence. Red solid line indicates predicted n-terminal region, orange solid line indicates the predicted center hydrophobic region, and yellow solid line indicates predicted c-terminal region of signal peptide. Black dotted line indicates the cleavage site (CS) of the signal peptide. Sec/SPI: Sec translocon transported secretory signal peptide/Signal Peptidase I Tat/SPI: Tat translocon transported Tat signal peptides/Signal Peptidase I (B) Western blot analysis demonstrating the presence of Toll-9/V5 in endosomes. Endosomal fractions were identified using Rab5 as a microsomal marker, while Actin served as a cytosolic marker. Data are representative of three independent experiments. (C) Micrographs show colocalization of Rab5-early endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (D) Micrographs show colocalization of Rab7-Late endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (E) Micrographs show colocalization of Rab5-early endosome marker (green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (F) Toll-9 (anti-V5 tag ab-Green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (G) Western blot analysis using the indicated antibodies following immunoprecipitation of V5 tag (Toll-9) using J2 dsRNA antibody from the lysate of Poly (I:C) treated Toll-9 OE and S2 cells in presence and absence of CuSO 4 (500 µM). Data are representative from three independent experiments.

Article Snippet: The cells were blocked in phosphate-buffered saline (PBS) containing 10% FBS and incubated with antibodies against Rab5 (1:50; Abcam ab31261), Rab7(1:20; Developmental Studies Hybridoma Bank Rab7), DCV Capsid (1:200; Abcam ab92954), dsRNA J2 (1:200; Jena Biosciences RNT-SCI-10010200) and V5 tag (1:200; ThermoFisher Scientific R960-25) for overnight at 4°C.

Techniques: In Silico, Sequencing, Western Blot, Marker, Immunoprecipitation

Journal: Oncogene

Article Title: Inhibition of E2F-4/DP-1-stimulated transcription by p202.

doi: 10.1038/sj.onc.1201184

Figure Lengend Snippet: Figure 4 Interaction of p202 with p107 and p130 in vitro and in vivo. (a) Binding of p202 translated in vitro to p107 linked to GST. 35S-labeled p202 was incubated with glutathione-Sepharose beads loaded with GST (lane 2), GST-p107 (lane 3), or GST-E2F-1 (lane 4). The bound proteins were eluted and analysed by SDS ± PAGE and ¯uorography. An aliquot of 35S-labeled p202 was also run (lane 1). The p202 band is indicated by an arrow. (b) Binding of p107 or p130 translated in vitro to p202 linked to GST. 35S-labeled p107 was incubated with glutathione-Sepharose beads loaded with GST (lane 2), GST-p202 (lane 3), or GST-E1A (lane 4). Similarly, 35S-labeled p130 was incubated with glutathione-Sepharose beads loaded with GST (lane 6), GST-p202 (lane 7), or GST- E1A (lane 8). The bound proteins were eluted and analysed by SDS ± PAGE and ¯uorography. An aliquot of 35S-labeled p107 (lane 1) or p130 (lane 5) was also run. The p107 and p130 bands are indicated by a long and a short arrow, respectively. (c) Binding of p107 in cell extracts to GST-202. Extracts from growing AKR-2B cells were incubated with glutathione-Sepharose beads loaded with GST (lane 2) or GST-202 (lane 3). After the beads were washed, the bound proteins were analysed by SDS ± PAGE and Western blotting with an anti-p107 monoclonal antibody. As a control, extracts were also immunoprecipitated with anti-p107 and the immunoprecipitates were run in lane 1. The p107 band is indicated by an arrow. (d) Binding of p130 in cell extracts to GST- p202. Extracts from growing AKR-2B cells were incubated with glutathione-Sepharose beads loaded with GST (lane 2) or GST- p202 (lane 3) or GST-E1A (lane 4). After the beads were washed, the bound proteins were eluted and analysed by SDS ± PAGE followed by Western blotting using anti-p130 antiserum. As a control extracts were also immunoprecipitated with anti-p130 and the immunoprecipitates were run in lane 1. The p130 band is indicated by an arrow. (e) Binding of labeled p107 or p130 translated in vitro to p202 in far-Western assay. Prestained protein markers (lanes 1, 4, and 7), GST (lanes 2, 5, and 8) and GST-p202 (19-445) (lanes 3, 6, and 9) were subjected to SDS ± PAGE and proteins were transferred to nitrocellulose membrane. Lanes 1 to 3 were stained for proteins with Ponceu stain, lanes 4 to 6 were probed with in vitro-translated 35S-methionine-labeled p107, and lanes 7 to 9 were probed with in vitro-translated 35S-methionine-labeled p130. The position of the GST-p202 band is indicated by an arrow. (f) Binding of p107 in cell extracts to p202 and its segments linked to GST. Upper panel: Extracts prepared from growing AKR-2B cells were incubated with gluthathione-Sepharose beads loaded with GST (lane 1), GST-p202 (19-445) (lane 3), GST-p202 (58-291) (lane 4), GST-p202 (255-293) (lane 5), or GST-p202 (295-445) (lane 6). As a positive control, extracts were also incubated with GST- E1A (lane 2). The bound proteins were eluted and analysed by SDS ± PAGE and immunoblotting using anti-p107. The p107 band is indicated by an arrow. Lower panel: schematic representation of the p202 segments whose binding to the cellular p107 was tested. The thin vertical lines indicate the borders of the 200-amino-acid repeat regions a and b. The numbers of the N- and C-terminal animoacyl residues of these sequences are indicated; binding: strong, ++; weak, +; undetectable 7. (g) Coimmunoprecipitation of p202 with p107. 35S-methionine-labeled extracts from AKR-2B cells untreated (lane 1) or treated with IFN (lane 2) were subjected to immunoprecipitations using anti-p107 antibodies. After being washed, the immunoprecipitates were analysed by SDS ± PAGE followed by ¯uorography. As a size marker, an aliquot of labeled p202 translated in vitro was also run (IVT-202, lane 3). The position of the p202 protein is indicated by an arrow. (h) Coimmunoprecipitation of p202 with p130. Extracts from control (lane 1) and IFN-treated AKR-2B cells (lane 2) were immunoprecipitated with anti-p130 antibodies and analysed by immunoblotting using anti-p202 conjugated to horseradish peroxidase. As a size marker, an aliquot of extract from IFN-treated cells was also run in lane 3. The position of the p202 band is indicated by an arrow. For further details see Materials and methods

Article Snippet: The extracts were incubated with anti-p107 (clone SD9) or anti-p130 (clone C-20) (Santa Cruz Biotechnology, Inc.) at 48C for 90 min.

Techniques: In Vitro, In Vivo, Binding Assay, Labeling, Incubation, SDS Page, Western Blot, Control, Immunoprecipitation, Membrane, Staining, Positive Control, Marker