Journal: Journal of Virology

Article Title: Adenovirus Modulates Toll-Like Receptor 4 Signaling by Reprogramming ORP1L-VAP Protein Contacts for Cholesterol Transport from Endosomes to the Endoplasmic Reticulum

doi: 10.1128/JVI.01904-16

Figure Lengend Snippet: Description of antibodies used in the study a

Article Snippet: Antibodies and reagents used in the study are described in and , respectively. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Target Source Vendor Catalog no. Actin Rabbit Sigma (St. Louis, MO) A2066 EGFR Rabbit Fitzgerald (Concord, MA) 20R-ER004 FLAG Mouse Sigma F3165 GFP Mouse Stressgen (San Diego, CA) SAB-500 HRS Rabbit Bethyl (Montgomery, TX) A300-989A Lamin B1 Rabbit Abcam (Cambridge, MA) ab16048 LAMP1 Mouse DSHB b H4A3 LAMP2 Mouse DSHB H4B NF-κB (p65) Rabbit Cell Signaling (Beverly, MA) 8242 NF-κB (pSer536) Rabbit Cell Signaling 3033 ORP1L Mouse Santa Cruz (San Diego, CA) sc-376602 ORP1L Rabbit Abcam ab203352 ORP5 Rabbit Novus (Littleton, CO) NBP2-19683 Rab5 Mouse BD Biosciences (San Jose, CA) 610281 Rab7 Rabbit Santa Cruz sc6563 SREBP1 Mouse Novus NB600-582 TLR4 Mouse R&D Systems (Minneapolis, MN) MAB14782 TfR Mouse Zymed (South San Francisco, CA) 13-6800 Tubulin Rabbit Cell Signaling 2125 VAP-A Rabbit Sigma HPA009174 VAP-B Mouse Proteintech (Rosemont, IL) 14477-1-AP Open in a separate window a Antibodies were routinely used at a dilution of 1:1,000 to 1:2,000 for immunoblotting and at a dilution of 1:100 to 1:500 for immunostaining. b DSHB, Developmental Studies Hybridoma Bank (developed under the auspices of the Eunice Kennedy Shriver National Institute of Child Health and Human Development [NICHD] and maintained by The University of Iowa Department of Biological Sciences [Iowa City, IA]).

Techniques:

Journal: Frontiers in immunology

Article Title: Structural and functional analysis of the small GTPase ARF1 reveals a pivotal role of its GTP-binding domain in controlling of the generation of viral inclusion bodies and replication of grass carp reovirus.

doi: 10.3389/fimmu.2022.956587

Figure Lengend Snippet: FIGURE 2 The subcellular co-localizations or interactions between gcARF1 and GCRV proteins. (A) The subcellular co-localizations between gcARF1 and GCRV proteins. CIK cells plated onto coverslips in 24-well plates were transfected with FLAG-tagged gcARF1. Then the cells were infected with the GCRV at an MOI of 1 or left untreated for another 24 h. Finally, the cells were washed and fixed with 4% PFA for immunofluorescence assays. The images were obtained by Leica confocal microscopy. Scale bars, 10 µm. (B) The interactions between gcARF1 and NS80, NS38, VP3 or VP5. CIK cells seeded in 10-cm2 dishes were transfected with indicated plasmids. After 24 h later, the cells were infected with the GCRV at an MOI of 1 or left untreated for another 24 h. Finally, the cells were harvested and used for protein extraction. Co-IP was performed with anti- FLAG-conjugated agarose beads. The cell lysates and bound proteins were analyzed by immunoblotting with the indicated Abs.

Article Snippet: The anti-FLAG mouse monoclonal antibody (#F3165), antipTurboGFP rabbit polyclonal antibody (#AB513) and antiGAPDH mouse monoclonal antibody (#60004-1-Ig) were purchased from Sigma-Aldrich, Everogen and Proteintech, respectively.

Techniques: Transfection, Infection, Confocal Microscopy, Protein Extraction, Co-Immunoprecipitation Assay, Western Blot

Journal: Frontiers in immunology

Article Title: Structural and functional analysis of the small GTPase ARF1 reveals a pivotal role of its GTP-binding domain in controlling of the generation of viral inclusion bodies and replication of grass carp reovirus.

doi: 10.3389/fimmu.2022.956587

Figure Lengend Snippet: FIGURE 7 The 27AAGKTT32 motif and T31 residue are required for the function of gcARF1 in promoting GCRV replication and infection. (A) Schematic representation of the gcARF1 and its mutants. (B) Crystal violet staining and determination of GCRV titers for overexpression of gcARF1 mutants including gcARF1(d27-32aa) and gcARF1(T31N) in CIK cells that were infected with GCRV at an MOI of 1 for 24 h. The asterisk above the error bars indicated statistical significance using the group transfected with FLAG as the control group. The asterisk above the bracket indicated statistical significance between the two groups connected by the bracket. (C) IB analysis of VP3, VP5, NS80 and NS38 proteins regulated by overexpression of gcARF1 or gcARF1 mutants including gcARF1(d27-32aa) and gcARF1(T31N) in CIK cells infected with GCRV. CIK cells seeded overnight in 12-well or 6-well plates were transiently transfected with indicated plasmids. After 24 h later, the cells were infected with the GCRV at an MOI of 1 or left untreated. The supernatants in 12-well plates were collected at 24 hpi for viral titer assays, and the cells were fixed and stained with crystal violet (B). The cells in 6-well plates were collected at 24 hpi for protein extraction (C). +: 500 ng, ++: 1000 ng. The expression ratios for viral proteins were quantified by Quantity One. (D) The interactions between gcARF1, gcARF1(d27-32aa) or gcARF1(T31N) and viral proteins. CIK cells seeded in 10-cm2 dishes were transfected with the indicated plasmids. After 24 h later, the cells were infected with or without the GCRV at an MOI of 1. Then the cells were harvested and lysed at 24 hpi. Co-IP was performed with anti-FLAG-conjugated agarose beads. The cell lysates and bound proteins were analyzed by immunoblotting with the indicated Abs.

Article Snippet: The anti-FLAG mouse monoclonal antibody (#F3165), antipTurboGFP rabbit polyclonal antibody (#AB513) and antiGAPDH mouse monoclonal antibody (#60004-1-Ig) were purchased from Sigma-Aldrich, Everogen and Proteintech, respectively.

Techniques: Residue, Infection, Staining, Over Expression, Transfection, Control, Protein Extraction, Expressing, Co-Immunoprecipitation Assay, Western Blot