Journal: PLoS ONE

Article Title: Inflammatory PAF Receptor Signaling Initiates Hedgehog Signaling and Kidney Fibrogenesis During Ethanol Consumption

doi: 10.1371/journal.pone.0145691

Figure Lengend Snippet: Chronic ethanol ingestion is fibrogenic in rat kidney. A) Fibrotic protein expression in kidney. Rats were fed the standard Lieber-deCarli liquid ethanol diet, or its isomaltose control, for 28 days before kidneys were excised, perfused with saline, fixed and sectioned as described in “Methods.” Sections were rehydrated and stained with anti-TGF-ß1, anti- α-smooth muscle actin, anti-collagen IV or anti-collagen 1 antibodies, and SP-streptavidin-conjugated secondary antibody for anti-TGF-ß1, or fluorescent secondary antibodies for the other sections as stated in “Methods.” TGF-ß sections were developed with horseradish peroxidase and DAB/metal chromogenic solution. B) mRNAs encoding fibrotic proteins were induced in kidneys of rats ingesting ethanol. Total RNA was isolated from ethanol and pair-fed kidneys from rats at the end of the feeding trial, and mRNA was quantified by SYBR Green one-step Reverse Transcription-PCR for the stated mRNAs and ribosomal 18S with the Bio-Rad MyiQ real-time PCR detection system. mRNA expression was normalized to 18S RNA content and 2 -ΔΔCT was used to calculate the fold changes. Data are expressed as mean±SEM (n = 4), and p

Article Snippet: The conjugates were then ligated by horseradish peroxidase-conjugated anti-mouse (1:10000) or anti-rabbit (1:5000) antibody before detection with Amersham Biosciences ECL Prime.

Techniques: Expressing, Staining, Isolation, SYBR Green Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Journal of Virology

Article Title: Immobilized Cobalt Affinity Chromatography Provides a Novel, Efficient Method for Herpes Simplex Virus Type 1 Gene Vector Purification

doi: 10.1128/JVI.78.17.8994-9006.2004

Figure Lengend Snippet: Western blot analysis of HAT-tagged gB. Protein lysates from Vero cells infected with KgBHAT (lanes 2 and 4) and the control virus KgBpK − (lanes 1 and 3) were separated by using the NuPAGE system and were transferred to a polyvinylidene difluoride membrane. The membrane was then cut in half, and each part was probed separately either with a monoclonal antibody against HSV gB (lanes 1 and 2) or with a polyclonal antibody against the HAT peptide (lanes 3 and 4), followed by detection with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody, respectively. M, molecular size marker; Ab, antibody.

Article Snippet: The membranes were then washed with PBS containing 0.05% Tween 20 (Sigma, St. Louis, Mo.) and incubated with either anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Sigma) in 2% milk-PBS for 1 h at 25°C.

Techniques: Western Blot, HAT Assay, Infection, Marker

Journal: Plant Physiology

Article Title: Exposed Loop Domains of Complexed 14-3-3 Proteins Contribute to Structural Diversity and Functional Specificity 1

doi: 10.1104/pp.105.073916

Figure Lengend Snippet: Identification of 14-3-3 isoforms recognized by three mAbs generated from 14-3-3/G-box DNA-binding complexes using 13 recombinant Arabidopsis 14-3-3 proteins. A, GBC mAbs detect a subset of nondenatured recombinant Arabidopsis 14-3-3 proteins. Samples of each purified recombinant Arabidopsis 14-3-3 protein were transferred to nitrocellulose with a dot-blot apparatus. After blocking, the blot was probed with GBC mAb 2A3, 4B9, and 5D6. Cross-reactivity was detected with anti-mouse horseradish peroxidase conjugate and chemiluminescence. B, GBC mAbs detect a subset of denatured recombinant Arabidopsis 14-3-3 proteins. Aliquots of 10 μ g of each recombinant Arabidopsis 14-3-3 proteins were denatured in reducing SDS sample buffer prior to electrophoresis. After transferring electrophoresed proteins to nitrocellulose and blocking, the blot was probed with GBC mAb 2A3, 4B9, and 5D6. Immuno-cross-reactivity was detected with anti-mouse horseradish peroxidase conjugate and chemiluminescence.

Article Snippet: Anti-rabbit and anti-mouse horseradish peroxidase conjugates were purchased from Amersham (Amersham Biosciences).

Techniques: Generated, Binding Assay, Recombinant, Purification, Dot Blot, Blocking Assay, Electrophoresis, Transferring