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Co-immune precipitation of Sdc-1 and Topors from lysates of NMuMG cells.

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posted on 17.08.2012, 00:17 by Kathleen R. Braun, Allison M. DeWispelaere, Steven L. Bressler, Nozomi Fukai, Richard D. Kenagy, Lihua Chen, Alexander W. Clowes, Michael G. Kinsella

(A) Total cell lysates prepared from NMuMG cells were immune precipitated (IP) with non-immune rat IgG (lane 1) or rat anti-Sdc-1 antibody (lane 2 and 3) and then western blotted for Sdc-1 (lanes 1–2), and after stripping, re-probed with anti-Topors (lanes 3). Total cell lysate (lane 4) was included as a control for anti-Topors immunoreactivity. Arrows mark major co-precipitated Topors bands (110 and 175 kDa). (B) Non-immune chicken IgY-preadsorbed total cell lysates of NMuMG cells were immunoprecipitated with chicken anti-Topors, and immune precipitates were heparinase- and chondroitin ABC lyase-digested prior to SDS-PAGE and western blotting. Lysate (lane 1) and Topors immunoprecipitates (lane 2 and 3) that were probed on western blots for Topors (lanes 1 and 2) had Topors bands (110 and 175 kDa bands, double arrow), or for Sdc-1 (lane 3), which gave a band of ∼85 kDa (large arrow, lane 3 and 4), similar in size to a band present in a Sdc-1-positive control (lane 4), prepared by ion-exchange chromatography from the medium of NMuMG cultures. Lower MW heavy bands represent chicken IgY (NChIgY) from the immune precipitate (arrow). Lanes 1–3 are from the same blot with lane 3 representing a stripped and re-probed lane of the Topors immune precipitate run in parallel to that shown in lane 2. Lane 4 represents an adjacent lane from the same blot, at a longer exposure time.

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