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. 2012 Nov 30;7(11):e50879. doi: 10.1371/journal.pone.0050879

Figure 1. Isolation and identification of ERp57 as a GDP-RalA binding protein.

Figure 1

(A) GST-RalA loaded with GDP or GTP was used as bait for pull-down experiments from the indicated rat tissue extracts. Bound proteins were analysed by SDS-PAGE and Coomassie blue staining. A GDP-dependent RalA binding protein, p58, is indicated. (B) MALDI-TOF mass spectrum of tryptic peptides derived from brain p58, showing peak identities, matched against theoretical digests with a mass accuracy of ±0.1 Da. (C) Western blotting of GST-RalA pull-down experiments from synaptosomes, whole brain or testis extracts, probed with anti-ERp57 or anti-RalBP1 antibodies, as indicated. (D) A whole testis lysate and a GST-RalA pull-down from testis extract were analysed by Western blot, probed with antibodies recognising ERp57, RalBP1 and PDI. Note that ERp57 was not clearly detected in the lysate in this experiment, due to the weak ERp57 antibody. Detection in pull-down assays was attributed to enrichment of ERp57 in these samples. In our subsequent work, we generated and used a stronger ERp57 antibody, which detects ERp57 in tissue extract (e.g., Fig. 3A).