Xenbase Image ID: 151887

Fig. 6. Characterization of antibodies. (A) Immunoblots of total protein extracts from stage 35 embryos were incubated with either the complete anti-a3 immune sera (D3F) or affinity purified antibody (D3FAP). XTC cells were first immunoprecipitated with the anti-b1 McAb 8C8, subjected to SDS-PAGE under reducing (+) and non-reducing (-) conditions and then immunoblotted. D3FAP recognizes 135–140 kDa doublet a3 bands from the total stage 35 embryo extract and a single 140 kDa a3 band in the b1 immunoprecipitate from XTC cells (non-reduced). The D3FAP immunoreactive C-terminal fragment of a3 runs off the 7% gel under reducing conditions (XTC + DTT lane). (B) Immunoblots of stage 45 embryo total protein extracts incubated with complete anti-Xenopus a2 immune sera (D5F) or affinity purified antibodies (D5FAP). The D5FAP antibody recognizes a single a2 protein of 160 kDa, which is not cleaved upon reduction (-). (C) Immunoblots of XTC cell extracts immunoprecipitated with D3FAP and incubated with either McAb 2F10 or 8C8. (D) XTC cell immunoblot of D3FAP immunoprecipitated material (1) or D3FAP immunodepleted extract (2). 2F10 recognizes the 140 kDa a3 band from the immunoprecipitate but not the immunodepleted supernatant. (E) Western blots of Xenopus a3 transfected (T) and non-transfected (N) mouse NIH 3T3 cells. 2F10 and D3FAP recognize the 140 kDa a3 protein in only the transfected cells. The anti-b1 antibody 363 blot is included as a loading control. All gel samples were prepared either in the presence (+) or absence (-) of dithiothreitol (DTT). Image published in: Meng F et al. (1997) Copyright © 1997. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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