Journal: Viruses

Article Title: Transcriptome and Proteomic Analysis Reveals Up-Regulation of Innate Immunity-Related Genes Expression in Caprine Herpesvirus 1 Infected Madin Darby Bovine Kidney Cells

doi: 10.3390/v13071293

Figure Lengend Snippet: Up-regulated targets in this study.

Article Snippet: The membrane was treated sequentially with 5% skimmed milk in TBS containing 0.5% Tween 20 (TBST) at 37 °C for 2 h, with different primary antibodies (1/200 diluted rabbit anti-Viperin polyclonal antibody (Abcam, Cambridge, UK), 1/200 diluted mouse anti-ISG15 antibody (Santa Cruz, Dallas, TX, USA, provided by Pfo.

Techniques: Multiplex sample analysis

Journal: Viruses

Article Title: Transcriptome and Proteomic Analysis Reveals Up-Regulation of Innate Immunity-Related Genes Expression in Caprine Herpesvirus 1 Infected Madin Darby Bovine Kidney Cells

doi: 10.3390/v13071293

Figure Lengend Snippet: Partial validation of RNA-Sequencing (RNA-Seq) and isobaric tags for relative and absolute quantitation (iTRAQ) data by Western blot. ( A ) The expression of RSAD2 (Viperin) in the samples was determined by Western blot. The molecular weight of Viperin was 42 kDa. Lane M, prestained protein mass markers; Lane 1, CpHV-1-infected MDBK cell sample; Lane 2, mock-infected MDBK cell sample. β-actin was the internal control; molecular weight was 42 kDa. ( B ) ISG15 western blot analysis of MDBK cells. The molecular weight of ISG15 was 15 kDa. Lane M, prestained protein mass markers; Lane 1, CpHV-1-infected MDBK cell sample; Lane 2, mock-infected MDBK cell sample. β-actin was the internal control. ( C ) MX1 western blot analysis of MDBK cells. The molecular weight of MX1 was 74.8 kDa. Lane M, prestained protein mass markers; Lane 1, CpHV-1-infected MDBK cell sample; Lane 2, mock-infected MDBK cell sample. β-actin was the internal control. The integrated density of every group was compared with that of internal loading control β-actin before the relative integrated density values between every infected group and its corresponding control group were calculated. Western strips were performed by grayscale analysis using the ImageJ software, and error bars represent relative integrated density mean standard deviations among three independent replicates. * p < 0.05 and ** p < 0.01.

Article Snippet: The membrane was treated sequentially with 5% skimmed milk in TBS containing 0.5% Tween 20 (TBST) at 37 °C for 2 h, with different primary antibodies (1/200 diluted rabbit anti-Viperin polyclonal antibody (Abcam, Cambridge, UK), 1/200 diluted mouse anti-ISG15 antibody (Santa Cruz, Dallas, TX, USA, provided by Pfo.

Techniques: Biomarker Discovery, RNA Sequencing, Quantitation Assay, Multiplex sample analysis, Western Blot, Expressing, Molecular Weight, Infection, Control, Software

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Expression tests for ejection proteins gp14, gp15, and gp16 (A) Diagram of plasmids for gp14, gp15, and gp16. (B) Diagram of transformed colonies in different expression strains growing on agar plates treated with ampicillin (100 μg/mL). (C) Diagram of 100 mL starter cultures treated with ampicillin for different expression strains in 250 mL Erlenmeyer flasks. (D) Diagram of 1 L LB cultures treated with ampicillin and inoculated 1:50 with starter culture for different expression strains. (E–G) SDS-PAGE analysis for gp14, gp15, and gp16 before/after (−/+) induction with IPTG at OD600 = 0.6 and expression. Expected molecular weights are gp14, 21 kDa, gp15, 87 kDa and gp16, 146 kDa as incdicated on the SDS-PAGE gels in cyan, magenta, and green, respectively.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Expressing, Transformation Assay, SDS Page

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Workflow for the reconstitution of gp15:gp16 periplasmic tunnel of the T7 DNA-ejectosome.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques:

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Purification of T7 ejection proteins gp15 and gp16 (A) SDS-PAGE results for sonicated samples for gp15 and gp16. Abbreviations: M = Markers for molecular weight, T = Total sample post-sonication, S = Supernatant sample, and P = Pellet sample after centrifugation. (B) SDS-PAGE results for Ni-NTA purification for gp15 and gp16. Abbreviations: Ft = Flow-through sample that passed through the gravity-flow column without binding, W 1 and W 2 = Wash 1 and 2 eluent samples, E n = Elution samples, and B = Beads sample representing protein still bound to the Ni beads. (C) Chromatograms and SDS-PAGE results for gel filtration purified gp15 and gp16 on a calibrated analytical Superdex200 10/300 column. Only relevant lanes from the same gel are shown. Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Purification, SDS Page, Sonication, Molecular Weight, Centrifugation, Binding Assay, Filtration, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Purification of T7 ejection protein gp14 (A) SDS-PAGE results for sonicated, NLS detergent extracted, and Ni-NTA purified samples of gp14. Abbreviations: M = Markers for molecular weight, T = Total sample post-sonication, S = Supernatant sample, and P = Pellet sample after centrifugation, US = Ultracentrifuged Supernatant sample, UP = Ultracentrifuged Pellet sample, Ft = Flow-through sample that passed through the gravity-flow column without binding, W 1 and W 2 = Wash 1 and 2 eluent samples, E n = Elution samples, and B = Beads sample representing protein still bound to the Ni beads. (B) Chromatogram and SDS-PAGE results for gel filtration purified gp14 solubilized in NLS (extraction) detergent. (C) Chromatogram and SDS-PAGE results for gp14 detergent exchange from NLS to DDM detergent using an anion exchange column. (D) Chromatogram and SDS-PAGE results for gel filtration purified gp15 solubilized in DDM (mild) detergent. Only relevant lanes from the same gel are shown in (B–D). Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Purification, SDS Page, Sonication, Molecular Weight, Centrifugation, Binding Assay, Filtration, Extraction, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: In vitro assembly of the T7 periplasmic tunnel from purified gp15 and gp16 (A) Diagram of complex components incubated in a 3:1 molar ratio on ice for 60 min (B) Chromatogram and SDS-PAGE analysis of gel filtrated gp15:gp16 sample. Magenta arrow represents gp15 expected molecular weight at 87 kDa. Green arrow represents gp16 expected molecular weight at 146 kDa. Only relevant lanes from the same gel are shown. Parts of this figure have been reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: In Vitro, Purification, Incubation, SDS Page, Molecular Weight, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet: Expected results for negative stain TEM and cryo-EM screening (A and B) Representative micrographs of (A) negatively stained and (B) vitrified gp15:gp16 complex. On the right-hand side are 2D-class averages. Scale bars, 100 nm. Reprinted from Molecular Cell, Vol 81, Issue 15, Swanson, et al., Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution, Pages 3145-3159.e7, Copyright (2021), with permission from Elsevier.

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Staining, Cryo-EM Sample Prep

Journal: STAR Protocols

Article Title: Expression and purification of phage T7 ejection proteins for cryo-EM analysis

doi: 10.1016/j.xpro.2021.100960

Figure Lengend Snippet:

Article Snippet: Optional: For further evidence of interaction between DNA-ejectosome components gp15 and gp16, run a NativePAGE (Invitrogen) with individual components versus the complex and observe shifts in band migration.

Techniques: Virus, Recombinant, Electron Microscopy, Filtration, Plasmid Preparation, Software, Chromatography, Electrophoresis, Microscopy

Journal: Scientific Reports

Article Title: Spautin-1 inhibits mitochondrial complex I and leads to suppression of the unfolded protein response and cell survival during glucose starvation

doi: 10.1038/s41598-022-15673-x

Figure Lengend Snippet: Autophagy inhibitors fail to show preferential cytotoxicity and suppress induction of ATF4 and XBP1s in glucose-starved or 2DG-stressed HT1080 cells. ( a ) HT1080 cells were treated with spautin-1 (10 μM) or SAR405 (1 μM) in HBSS containing HCQ (30 μM) for 4 h. Autophagosomes were visualized with the CYTO-ID Autophagy detection kit 2.0. ( b ) Effects of bafilomycin A1 (Baf), HCQ, and SAR405 on cell viability in GS-stressed HT1080 cells were determined by the CellTiter-Glo luminescent cell viability assay. Data are shown as mean ± SD ( n = 3). ( c ) Effects of spautin-1 (10 μM) and other autophagy inhibitors (Baf; 10 nM, HCQ; 30 μM, SAR405; 10 μM) on nuclear ATF4 and XBP1s induction under 2DG-stressed conditions were visualized using the Operetta CLS. Blue, red, and green fluorescent signals indicate nuclei, ATF4, and XBP1s, respectively. ( d ) Mean intensities of nuclear ATF4 and XBP1s intensities were determined using Harmony high-content analysis software. Data are shown as mean ± SD ( n = 3).

Article Snippet: Fluorescent images (nine fields per well) were acquired using a 20 × water objective lens by Operetta CLS (Perkin Elmer).

Techniques: Cell Viability Assay, High Content Screening, Software

Journal: Scientific Reports

Article Title: Spautin-1 inhibits mitochondrial complex I and leads to suppression of the unfolded protein response and cell survival during glucose starvation

doi: 10.1038/s41598-022-15673-x

Figure Lengend Snippet: USP10 and USP13 silencing has little effect on the UPR and cell viability under GS- or 2DG-stressed conditions. ( a ) Effects of USP10 and USP13 silencing on GRP78 in HT1080 cells were determined by western blotting. RPS3 was used as a loading control. The blot membranes were cut prior to hybridization with antibodies, according to Full range rainbow molecular weight markers. Original blots were presented in Supplementary Fig. . ( b ) Effects of USP10 and USP13 silencing on ATF4 and XBP1s induction in vehicle- or spautin-1-treated HT1080 cells under 2DG-stressed conditions were visualized using the Operetta CLS. ( c , d ) Mean intensities of nuclear ( c ) ATF4 and ( d ) XBP1s in ( b ) were determined using Harmony high-content analysis software. Data are shown as mean ± SD ( n = 3). ( e ) Effects of USP10 and USP13 silencing on cell viability under GS were determined by the CellTiter-Glo luminescent cell viability assay. Data are shown as mean ± SD ( n = 3). ( f ) Effects of USP10 and USP13 silencing on preferential cytotoxicity of spautin-1 under GS were determined by the CellTiter-Glo luminescent cell viability assay. Data are shown as mean ± SD ( n = 3).

Article Snippet: Fluorescent images (nine fields per well) were acquired using a 20 × water objective lens by Operetta CLS (Perkin Elmer).

Techniques: Western Blot, Control, Hybridization, Molecular Weight, High Content Screening, Software, Cell Viability Assay