Human autoantibodies against the SRP54G domain inhibit crosslinking of SRP54 to the PPL signal sequence. (a) Canine SRP was preincubated with no additions (lane 9), with IgG fractions containing anti-SRP autoantibodies (lanes 3 to 8), with affinity-purified autoantibodies (Aff, lanes 10 and 11) or with their accompanying unbound fractions (Sup, lanes 12 and 13), and subsequently incubated with a 35S-radiolabelled fragment of preprolactin (PPL) present in the form of a ribosome nascent chain complex (PPL86). The interaction of the SRP54 subunit with the signal sequence of PPL86 was determined by UV-induced crosslinking. A non-irradiated sample (lane 1) and a sample lacking exogenously added SRP (lane 2) are shown as controls. The ~63 kDa crosslinking product (PPL86/SRP54) resulting from the crosslinking of the 86-residue PPL fragment to the 54 kDa subunit of canine SRP is indicated by a white arrowhead. A faint ~61 kDa product is visible in some lanes, especially where the binding of the canine SRP is strongly inhibited. This smaller species represents crosslinking of the nascent chain to the endogenous wheatgerm SRP54 homologue, and this product is normally only observed in the absence of canine SRP . The ~9 kDa PPL86 fragment is indicated, as are the locations of full-length preprolactin (**) resulting from incomplete linearization of the DNA template for transcription of PPL86, and a peptidyl-tRNA species (*) resulting from the incomplete hydrolysis of the PPL86-tRNA bond during sample preparation. (b) The intensity of the 63 kDa band in (a) was quantified by scanning with an LKB Ultroscan XL enhanced laser densitometer. The most intense band achieved with a control serum or blank in each set (for instance, lanes 3–8 and lanes 9–13) was set to 100%.