glutathione sepharose 4 fast flow  (GE Healthcare)

Journal: Journal of Lipid Research

Article Title: Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding

doi: 10.1194/jlr.M041129

Figure Lengend Snippet: Binding of PCSK9 to recombinant EGF-A. A: Purification of GST:EGF-A fusion protein. GST:EGF-A fusion protein was purified using Glutathione Sepharose 4 Fast Flow affinity gel chromatography, followed by size-exclusion chromatography on a Tricorn Superose

Article Snippet: The GST:EGF-A fusion protein was purified using Glutathione Sepharose 4 Fast Flow (GE Healthcare) affinity gel chromatography according to the manufacturer's protocol.

Techniques: Binding Assay, Recombinant, Purification, Flow Cytometry, Chromatography, Size-exclusion Chromatography

Journal: PLoS Pathogens

Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

doi: 10.1371/journal.ppat.1006851

Figure Lengend Snippet: NP interacts with PLSCR1 in mammalian cells. (A, B) Co-IP assay of V5-NP and Flag-PLSCR1 in HEK293T cells. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Flag mAb (B) and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (C, D) GST pull-down assay of NP and PLSCR1. Lysates of HEK293T cells transfected with the GST or GST-PLSCR1 construct were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-WSNNP (C); HEK293T cell lysates containing exogenously expressed GST or GST-WSNNP were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-PLSCR1 (D). After washing away the unbound proteins, equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by western blotting using a rabbit anti-NP pAb, a rabbit anti-GST pAb, or a rabbit anti-PLSCR1 pAb. GST, GST-PLSCR1, or GST-WSNNP proteins in the eluates were detected by Coomassie blue (CB) staining. (E) The NP-PLSCR1 interaction does not rely on RNA binding. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (F) PLSCR1 interacts with NP during natural viral infection. Confluent A549 cells grown in 6-well plates were mock infected with PBS or infected with WSN virus at an MOI of 5. At 6 h p.i., cell lysates were immunoprecipitated with a rabbit anti-PLSCR1 pAb and were subjected to western blotting with a mouse anti-NP mAb or a rabbit anti-PLSCR1 pAb to detect NP and PLSCR1, respectively. (G) Mapping of the PLSCR1-interacting domain within NP. Schematic presentation of influenza NP showing the different domains as well as the truncation mutants made in this study is on the left side. The interaction between PLSCR1 and the NP truncation mutants is shown on the right side. Lysates of HEK293T cells were pulled down with glutathione magnetic beads. The bound proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-GST pAb to reveal the presence of PLSCR1 and NP, respectively. NES, nuclear export signal; NAS, nuclear accumulation signal.

Article Snippet: Then, 300 μl of the cleared lysates from cells transfected with pCAGGS-GST, pCAGGS-GST-PLSCR1, or pCAGGS-GST-NP was mixed with 40 μl of Glutathione Sepharose 4 Fast Flow (GE Healthcare, Pittsburgh, PA) and rocked for 1 h at 4°C.

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Construct, Incubation, Flow Cytometry, Staining, RNA Binding Assay, Infection, Magnetic Beads

Journal: PLoS Pathogens

Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

doi: 10.1371/journal.ppat.1006851

Figure Lengend Snippet: NP interacts with PLSCR1 in mammalian cells. (A, B) Co-IP assay of V5-NP and Flag-PLSCR1 in HEK293T cells. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Flag mAb (B) and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (C, D) GST pull-down assay of NP and PLSCR1. Lysates of HEK293T cells transfected with the GST or GST-PLSCR1 construct were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-WSNNP (C); HEK293T cell lysates containing exogenously expressed GST or GST-WSNNP were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-PLSCR1 (D). After washing away the unbound proteins, equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by western blotting using a rabbit anti-NP pAb, a rabbit anti-GST pAb, or a rabbit anti-PLSCR1 pAb. GST, GST-PLSCR1, or GST-WSNNP proteins in the eluates were detected by Coomassie blue (CB) staining. (E) The NP-PLSCR1 interaction does not rely on RNA binding. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (F) PLSCR1 interacts with NP during natural viral infection. Confluent A549 cells grown in 6-well plates were mock infected with PBS or infected with WSN virus at an MOI of 5. At 6 h p.i., cell lysates were immunoprecipitated with a rabbit anti-PLSCR1 pAb and were subjected to western blotting with a mouse anti-NP mAb or a rabbit anti-PLSCR1 pAb to detect NP and PLSCR1, respectively. (G) Mapping of the PLSCR1-interacting domain within NP. Schematic presentation of influenza NP showing the different domains as well as the truncation mutants made in this study is on the left side. The interaction between PLSCR1 and the NP truncation mutants is shown on the right side. Lysates of HEK293T cells were pulled down with glutathione magnetic beads. The bound proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-GST pAb to reveal the presence of PLSCR1 and NP, respectively. NES, nuclear export signal; NAS, nuclear accumulation signal.

Article Snippet: Then, 300 μl of the cleared lysates from cells transfected with pCAGGS-GST, pCAGGS-GST-PLSCR1, or pCAGGS-GST-NP was mixed with 40 μl of Glutathione Sepharose 4 Fast Flow (GE Healthcare, Pittsburgh, PA) and rocked for 1 h at 4°C.

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Construct, Incubation, Flow Cytometry, Staining, RNA Binding Assay, Infection, Magnetic Beads

Journal: Scientific Reports

Article Title: Human Heat shock protein 40 (Hsp40/DnaJB1) promotes influenza A virus replication by assisting nuclear import of viral ribonucleoproteins

doi: 10.1038/srep19063

Figure Lengend Snippet: Hsp40 interacts with incoming viral ribonucleoprotein complexes in IAV infected cells. ( a ) Hsp40 interacts with NP component of vRNPs. A549 cells were infected with PR8 virus at MOI = 10 in the presence of cycloheximide and cells were harvested 4 h post-infection. Cell extracts were immunoprecipitated using anti-NP, anti-Hsp40, control IgG mouse and rabbit antibodies. RIP was analyzed with quantitative RT-PCR using NP vRNA specific primers. The fold change in NP vRNA pull down levels was calculated with reference to control antibodies IgG mouse and rabbit. ( b ) Cycloheximide (CHX) treatment significantly reduced NP mRNA and vRNA levels in infected cells. The input sample used to set up RNA immunoprecipitation was tested for NP levels in cycloheximide treated and untreated cells by qRT-PCR. The fold change in NP mRNA and vRNA levels was calculated with reference to CHX untreated cells. ( c ) RNase A treatment partially affects the interaction between NP and Hsp40. A549 cells were infected with virus at MOI = 1 and cells were harvested 24 h post-infection. Cell lysate was treated with RNase (100μg/ml) at 4 °C for 45 min followed by immunoprecipitation using anti-Hsp40 antibody. RNA was extracted from remaining cell lysate and was analyzed on formaldehyde agarose gel. ( d ) NP co-localizes with Hsp40 during early stages of infection. A549 cells were mock infected and infected with PR8 virus at MOI = 5 and were fixed at different time intervals. NP and Hsp40 were stained using anti-NP monoclonal antibody and anti-Hsp40 primary antibody, followed by Alexa 594 and Alexa 488 conjugated secondary antibodies respectively. Arrows indicate the co-localization of NP and Hsp40. ( e ) Hsp40 co-localizes with NP and vRNP. A549 cells were mock infected and infected with PR8 virus at MOI = 5 for 4 h and 24 h. NP and Hsp40 were stained using anti-NP antibody and anti-Hsp40 primary antibody followed by Alexa 350 and Alexa 488 conjugated secondary antibodies respectively. NP vRNAs were detected by positive sense RNA probes using FISH. Arrows indicate the co-localization of IAV NP vRNA, NP and Hsp40. Data show mean ± S.D. from one representative experiment (n = 3) of at least three independent experiments.

Article Snippet: For GST pull down, soluble fraction containing either GST or Hsp40-GST were incubated with glutathione sepharose beads (GST sepharose 4 Fast Flow, GE healthcare).

Techniques: Infection, Immunoprecipitation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Staining, Fluorescence In Situ Hybridization