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. 2007 Dec 10;36(2):648–665. doi: 10.1093/nar/gkm1045

Figure 8.

Figure 8.

The monoclonal anti-ORF2pM292–480 antibody 2H9 detects endogenous ORF2-encoded proteins. (A and B) Confocal images of HeLa cells transfected with either ORF2p-expressing pEF6-rORF2 (A) or the parental empty vector pEF6V5-His (B) and stained with the 2H9 IgM (red). (C) Immunoblot analysis examining the specificity of the generated monoclonal antiORF2p antibody. Fifty micrograms of whole cell extract from pEF6-rORF2 (lane 2)- or pEF6V5-His-transfected (lane 1) REF cells were loaded on a 6% SDS–PAA gel. In contrast to the parental empty expression vector pEF6V5-His, pEF6-rORF2 is expressing intact L1Rn ORF2p. As a loading control the membrane was stripped and incubated with an anti-α1-catenin antibody (lower panel). (D) Immunoblot analysis of RCL cell extract with monoclonal 2H9 antibody reveals two bands representing 150- and 90-kDa polypeptides. The 150-kDa band corresponds to the theoretical molecular mass of L1Rn ORF2p. (E) Silver staining of L1Rn ORF2-encoded proteins immunoprecipitated from RCL cell lysates with 2H9 IgM. Two polypeptides with apparent molecular masses of 150 and 90 kDa were detected and are consistent with the proteins detected by immunoblot analyses. Scale bar—10 µm.