Figure 5.
An in-frame region upstream of the 175-kDa cDNA encodes amino acid sequences present in the larger HARE subunits. LECs were lysed in Laemmli (1970) buffer containing 5% β-mercaptoethanol and samples were subjected to SDS-PAGE and electrotransfer. Nitrocellulose strips were cut and incubated with lane 1, a mixture of eight mAbs that recognize all three HARE proteins (i.e., the 175-kDa HARE and the 260- and 230-kDa subunits of the 300 HARE complex); lane 2, preimmune goat IgG; lane 3, goat IgG (Ab2 in the diagram) raised against a 16-amino acid putative coding region (TVLVPSRRAFEDMDQNK−91) upstream of the amino terminal start of the purified rat 175-kDa protein; lane 4, preimmune sheep IgG; and lane 5, sheep IgG (Ab1 in the diagram) raised against a peptide corresponding to the sequence PKCPLKSKGVKK773 within the rat 175-kDa protein. Strips were washed and incubated with the appropriate secondary antibody-alkaline phosphatase conjugates and substrates for color development.