Journal: International Journal of Molecular Sciences

Article Title: Recombinant Subunit Vaccine Candidate against the Bovine Viral Diarrhea Virus

doi: 10.3390/ijms25168734

Figure Lengend Snippet: Antibodies used for the detection of E2 proteins in Western blot assays.

Article Snippet: 1cE2-VSV-6xHis , Rabbit anti-VSV-G tag polyclonal antibody (cod. A190-131A. Bethyl laboratories. Inc, Montgomery, TX, USA) 1/5000 , Goat anti-Rabbit IgG (H+L) antibody Alexa Fluor ® 680 (Thermo Fischer Scientific, Waltham, MA, USA) 1/30,000 , Mouse anti-6xHis tag monoclonal antibody (cod. 631212, Clontech Laboratories, Waltham, MA, USA 1/5000 , Goat anti-Mouse IgG (H+L) antibody, Alexa FluorTM 790 (Thermo Fischer Scientific, Waltham, MA, USA) 1/30,000.

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: Retrograde Transport of Golgi-localized Proteins to the ER

doi:

Figure Lengend Snippet: Localization of VSVGtsO45 chimeras at nonpermissive (40°C) and permissive (32°C) temperatures. COS cells were transiently transfected with the indicated constructs and maintained at either 40° or 32°C. After 40 h, cells were fixed, permeabilized, and prepared for indirect immunofluorescence using polyclonal anti-VSV antibodies followed by rhodamine anti–rabbit secondary antibodies. At 40°C, VSVGtsO45, as well as each of the chimeras, were retained in the ER ( left ), whereas at 32°C, the constructs were distributed to the Golgi complex and/or the plasma membrane ( right ). β-COP staining ( bottom ) indicates the distribution of the Golgi complex, and was from cells double labeled for VSVG–Leu15. Bar, 10 μm.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-VSV antiserum (provided by C. Machamer, Johns Hopkins University, School of Medicine, Baltimore, MD); conformation-specific mouse monoclonal anti-VSVG antibodies (I1 [8G5F11] and I14 [1E9F9]; provided by D. Lyles, Wake Forest University, Winston-Salem, NC; ); rabbit polyclonal antibodies to mannosidase II (provided by K. Moreman); mouse monoclonal anti–mannosidase II antibody (53FC3; provided by B. Burke, University of Calgary, Calgary, Alberta, Canada); rabbit polyclonal anti-ribophorin antiserum (provided by G. Kreibich, New York University Medical Center, New York, NY); polyclonal antiserum versus ER membrane proteins (provided by D. Louvard, Institute Curie, Paris, France); rabbit polyclonal antiserum versus GM130 (provided by G. Warren, Imperial Cancer Research Fund, London, England); rabbit polyclonal antiserum versus furin (provided by E. Dell'Angelica, NIH, Bethesda, MD and J. Bonifacino); rabbit polyclonal anti–β-coat protein (COP) antiserum; and mouse monoclonal antibodies against epitopes in Myc (9E10; provided by J. Humphrey, NIH, Bethesda, MD) and HA (HA.11; from BAbCO, Richmond, CA).

Techniques: Transfection, Construct, Immunofluorescence, Clinical Proteomics, Membrane, Staining, Labeling

Journal: The Journal of Cell Biology

Article Title: Retrograde Transport of Golgi-localized Proteins to the ER

doi:

Figure Lengend Snippet: The relationship between misfolding and recycling in intact cells. COS cells expressing VSVG–TGN38 were pulse labeled at 40°C, and then chased in unlabeled medium at 32°C for either 5 min or 2 h. After each chase point, equal aliquots were shifted back to 40°C for an additional 10 min. Cells were solubilized and immunoprecipitated with polyclonal anti-VSV or monoclonal I14 antibodies. Whereas labeled material within the ER is sensitive to misfolding, material chased into the Golgi becomes resistant. Continued incubation at 40°C leads to a decrease in the percentage of folded but not total VSVG–TGN38. A subsequent shift back to 32°C permits refolding of VSVG–TGN38. Notice that a wild-type VSVG–TGN38 chimera is totally resistant to misfolding at 40°C. Quantitation reflects the average of two independent experiments.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-VSV antiserum (provided by C. Machamer, Johns Hopkins University, School of Medicine, Baltimore, MD); conformation-specific mouse monoclonal anti-VSVG antibodies (I1 [8G5F11] and I14 [1E9F9]; provided by D. Lyles, Wake Forest University, Winston-Salem, NC; ); rabbit polyclonal antibodies to mannosidase II (provided by K. Moreman); mouse monoclonal anti–mannosidase II antibody (53FC3; provided by B. Burke, University of Calgary, Calgary, Alberta, Canada); rabbit polyclonal anti-ribophorin antiserum (provided by G. Kreibich, New York University Medical Center, New York, NY); polyclonal antiserum versus ER membrane proteins (provided by D. Louvard, Institute Curie, Paris, France); rabbit polyclonal antiserum versus GM130 (provided by G. Warren, Imperial Cancer Research Fund, London, England); rabbit polyclonal antiserum versus furin (provided by E. Dell'Angelica, NIH, Bethesda, MD and J. Bonifacino); rabbit polyclonal anti–β-coat protein (COP) antiserum; and mouse monoclonal antibodies against epitopes in Myc (9E10; provided by J. Humphrey, NIH, Bethesda, MD) and HA (HA.11; from BAbCO, Richmond, CA).

Techniques: Expressing, Labeling, Immunoprecipitation, Incubation, Quantitation Assay

Journal: The Journal of Cell Biology

Article Title: Retrograde Transport of Golgi-localized Proteins to the ER

doi:

Figure Lengend Snippet: Fractionation of ER and Golgi membranes and misfolding in vitro. ( A ) COS cells transfected with VSVG–TGN38 were labeled with [ 35 S]methionine, chased to generate labeled molecules in both the ER and Golgi complex, and then homogenized and fractionated on a 0–26% Optiprep gradient, as described in Materials and Methods. Fractions were analyzed for the presence of specific markers; ribophorin ( ER ), and galactosyltransferase activity ( Golgi ). ( B ) Pooled ER and Golgi fractions were aliquoted into equal volumes and incubated at either 32° or 40°C for 60 min. Membranes were lysed at the indicated temperatures, placed on ice, and immunoprecipitated with polyclonal anti-VSV antiserum or conformation-sensitive anti-VSVG I14 antibodies. Precipitates were analyzed by SDS-PAGE and scanning densitometry. Notice the relative ability of I14 antibodies to recognize VSVG–TGN38 from Golgi, but not from ER, fractions at 40°C.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-VSV antiserum (provided by C. Machamer, Johns Hopkins University, School of Medicine, Baltimore, MD); conformation-specific mouse monoclonal anti-VSVG antibodies (I1 [8G5F11] and I14 [1E9F9]; provided by D. Lyles, Wake Forest University, Winston-Salem, NC; ); rabbit polyclonal antibodies to mannosidase II (provided by K. Moreman); mouse monoclonal anti–mannosidase II antibody (53FC3; provided by B. Burke, University of Calgary, Calgary, Alberta, Canada); rabbit polyclonal anti-ribophorin antiserum (provided by G. Kreibich, New York University Medical Center, New York, NY); polyclonal antiserum versus ER membrane proteins (provided by D. Louvard, Institute Curie, Paris, France); rabbit polyclonal antiserum versus GM130 (provided by G. Warren, Imperial Cancer Research Fund, London, England); rabbit polyclonal antiserum versus furin (provided by E. Dell'Angelica, NIH, Bethesda, MD and J. Bonifacino); rabbit polyclonal anti–β-coat protein (COP) antiserum; and mouse monoclonal antibodies against epitopes in Myc (9E10; provided by J. Humphrey, NIH, Bethesda, MD) and HA (HA.11; from BAbCO, Richmond, CA).

Techniques: Fractionation, In Vitro, Transfection, Labeling, Activity Assay, Incubation, Immunoprecipitation, SDS Page