Journal: PLoS ONE
Article Title: Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line
Figure Lengend Snippet: Identification of HSPGs expressed by a rat parathyroid cell line. A. RT-PCR analysis of PTr cells using syndecan-specific primers (see Materials and Methods for details). Total RNA was isolated from confluent cells and subjected to RT-PCR analysis. Amplified products were run on 2% agarose gel, stained with ethidium bromide and photographed under UV transilluminator. Lanes: M – 100 bp marker; SN1 – amplification with syndecan-1 specific primers; SN2 – amplification with syndecan-2 specific primers; SN3 – amplification with syndecan-3 specific primers; SN4 – amplification with syndecan-4 specific primers; G – amplification with GAPHD specific primers; (-) – negative controls containing no cDNA. B. Identification of HSPGs present in DRM fractions using WB analysis. Proteoglycans were isolated from confluent rat parathyroid cells and partially purified using Q-Sepharose anion-exchange chromatography. A proteoglycan-enriched fraction was incubated in the presence or absence of heparitinase I, subjected to SDS-PAGE and immunoblotted with anti-syndecan-1, anti-syndecan-4 or anti-ΔHS (3G10) antibodies. Lanes: 1, 4 and 7 represent the heparitinase I only; 2, 5 and 8 correspond to the control samples, incubated without heparitinase I; 3, 6, 8 correspond to the heparitinase-treated samples.
Article Snippet: Biotinylated mouse anti-ΔHS (3G10) antibodies, recognizing HS neo-epitope, generated by the digestion with heparitinase I from Flavobacterium heparinum and heparitinase I (Flavobacterium heparinum ) were purchased from Seikagaku Corporation, (Tokyo, Japan).
Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Agarose Gel Electrophoresis, Staining, Marker, Western Blot, Purification, Chromatography, Incubation, SDS Page