Coli Bl21, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 98/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 98 stars, based on 2 article reviews
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1) Product Images from "Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity"
Article Title: Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity
Journal: PLoS ONE
Figure Legend Snippet: Construction and detection of the CFA/I/II/IV-STa -toxoid -dmLT MEFA. (A) Construction of the CFA/I/II/IV MEFA. The most antigenic epitopes of the CS1, CS2, CS3, CS4, CS5 and CS6 major structural subunits were embedded into CFA/I major subunit by replacing the CfaB surface-exposed but less antigenic epitopes. (B) Construction of the 3xSTa N12S -dmLT toxoid fusion. Three copies of the STa toxoid STa N12S gene were genetically fused to the monomeric dmLT (LT R192G/L211A ) gene using SOE (splicing overlap extension) PCRs. (C) Construction of CFA/I/II/IV-STa N12S -dmLT MEFA. A substitution of the first 150 amino acids of the 3xSTa N12S -dmLT (the N-terminal STa N12S and the first 131 amino acids of LT-A subunit) with the CFA/I/II/IV MEFA created the CFA/I/II/IV-STa N12S -dmLT MEFA. Four linkers: LGA, GPVD, Gly-Pro linker GPGP, and L-linker were used for the construction. (D) Western blot to detect the CFA/I/II/IV-STa N12S -dmLT MEFA protein with anti-CFA/I, anti-CS1, -CS2, -CS3, -CS4, -CS5, and anti-CS6 MAb hybridoma supernatant (1:100; provided by Dr. AM Svennerholm), and rabbit anti-CT (1:3300; Sigma) and anti-STa antiserum (1:3300; provided by Dr. DC Robertson). Extracted MEFA proteins separated in 12% PAGE gel were detected with each anti-adhesin MAb, anti-CT and anti-STa antiserum and IRDye-labeled goat anti-mouse IgG or anti-rabbit IgG (1:5000; LI-COR). Lane (+) indicated the CFA/I/II/IV-STa N12S -dmLT MEFA proteins, whereas lane (-) of extracted total proteins of E . coli BL21 host strain as the negative control. Lane M is the protein marker (in kilo Daltons; Precision Plus Protein pre-stained standards; Bio-Rad).
Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis, Labeling, Negative Control, Marker, Staining
2) Product Images from "AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs"
Article Title: AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs
Journal: Frontiers in Plant Science
Figure Legend Snippet: Expression patterns and subcellular localization of AtPR5K2. (A) The expression of AtPR5K2 in various tissues of Arabidopsis thaliana . Total RNA was extracted from the roots, rosette leaves, cauline leaves, stems, flowers, and siliques of the wild-type plants. The transcript levels of AtPR5K2 were measured using quantitative reverse transcription PCR (qRT-PCR) and calculated relative to the expression of the endogenous control gene, TUBULIN2 . Error bars represent the ± SD from three independent experiments. (B) Subcellular localization of AtPR5K2 in Arabidopsis protoplasts. 35S:AtPR5K2-GFP and Aquaporin-RFP were coexpressed in Arabidopsis protoplasts, which were analyzed using confocal fluorescence microscopy and photographed after 24 h of incubation at 22°C. Aquaporin-RFP is a plasma-membrane marker. Scale bars represent 10 μm. (C) Subcellular localization of AtPR5K2 in the epidermal cells of tobacco ( Nicotiana benthamiana ) leave expressing 35S:AtPR5K2-GFP before and after plasmolysis. The epidermal cells were analyzed using confocal fluorescence microscopy and photographed after 48 h of incubation at 25°C. Scale bars represent 20 μm. Red asterisk indicates that AtPR5K2-GFP signal remains in the Hechtian strands. Red arrowheads point to the retracted plasma membrane. (D) In vitro kinase assays of AtPR5K2. The upper panel indicates the schematic structure of the GST-fused AtPR5K2 kinase domain (wPR5K2KD) and the GST-fused mutagenized AtPR5K2 kinase domain (mPR5K2KD). Each kinase domain was individually expressed in Escherichia coli , and 2 μg purified proteins was incubated in kinase assay buffer. Radioactive-labeled products were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and detected using radioactivity (bottom right). After electrophoresis, the purified products were stained with Coomassie brilliant blue (bottom left).
Techniques Used: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Fluorescence, Microscopy, Incubation, Marker, In Vitro, Purification, Kinase Assay, Labeling, Polyacrylamide Gel Electrophoresis, SDS Page, Radioactivity, Electrophoresis, Staining
3) Product Images from "PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence"
Article Title: PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence
Journal: PLoS Pathogens
Figure Legend Snippet: PIAS1 interacts with multiple proteins in the RNP complex of IAV. (A, B) Interaction of V5-WSNNP and Myc-PIAS1 in HEK293T cells by using a co-IP assay. HEK293T cells were individually transfected or co-transfected with plasmids expressing V5-WSNNP and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Myc mAb (B), and subjected to western blotting with a rabbit anti-V5 pAb and a rabbit anti-Myc pAb for the detection of WSNNP and PIAS1, respectively. (C) Interaction of GST-WSNNP and His-PIAS1 by using a GST pull-down assay. His-tagged PIAS1 was expressed in E . coli BL21 (DE3) and purified by using Ni Sepharose Excel resin, and the GST or GST-NP protein was expressed in HEK293T cells and purified by using Glutathione Sepharose 4 Fast Flow. An equal amount of purified PIAS1 was mixed with the Glutathione Sepharose 4 Fast Flow samples that bind GST or GST-NP. After rocking and washing, the mixed samples were separated by SDS-PAGE and stained with Coomassie blue. (D) Interaction of IAV NP and PIAS1 in virus-infected cells. HEK293T cells were transfected for 24 h to express Myc-PIAS1, and were then infected with WSN (H1N1) virus (MOI = 5). At 30 h p.i., cell lysates were immunoprecipitated with a mouse anti-NP mAb, followed by western blotting with a rabbit anti-NP pAb and a rabbit anti-Myc pAb. (E) Co-localization of IAV NP and PIAS1 in A549 cells infected with WSN (H1N1) virus. A549 cells were infected with WSN (H1N1) virus (MOI = 5). At 2, 4, 6, and 8 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb and a rabbit anti-PIAS1 pAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H+L) (red) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (green). The nuclei were stained with DAPI. (F-H) Co-IP assay to examine the interactions between Myc-PIAS1 and PB2, PB1, and PA of WSN (H1N1) virus in HEK293T cells. HEK293T cells were individually transfected or co-transfected with plasmids expressing WSNPB2, WSNPB1, WSNPA, and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-Myc mAb and were subjected to western blotting with a rabbit anti-PB2 pAb (F), a rabbit anti-PB1 pAb (G), a rabbit anti-PA pAb (H), and a rabbit anti-Myc pAb (F-H) for the detection of PB2, PB1, PA, and PIAS1, respectively.
Techniques Used: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Purification, SDS Page, Staining, Infection, Incubation