BNIP-H contains two Pin1-binding sites and interacts with the WW domain of Pin1.

<p>Purified GST fusion proteins of Pin1 full length, Pin1 WW domain and Pin1 PPI domain were incubated with PC12 whole cell lysates containing either HA-BNIP-H full length or various BNIP-H deletions proteins, which were expressed under NGF-stimulation (A, <i>in vitro</i> binding). After incubation, GST fusion proteins were isolated, washed and analyzed for the presence of bound BNIP-H or its mutants by western-blotting with anti-HA antibody. The membrane with the blotted proteins was than stained with amido black to reveal equal loading of GST and GST fusion proteins. GST protein was used as a control. Input shows approximately 2% of the lysate used for the GST pull-down assay. I, input; G, GST; P, Pin1 full length; WW, WW domain of Pin1; PPI, PPI domain of Pin1 (B). Various HA-tagged BNIP-H expression constructs (A, <i>in vivo</i> binding) and a FLAG-Pin1 expression plasmid were co-transfected into 293T cells. Lysates were subjected to immunoprecipitation with anti-FLAG antibody. Precipitates and whole cell lysates were analyzed by western-blotting with the indicated antibodies to detect binding of the different HA-BNIP-H fragments. A summary of the results is presented (A, <i>in vivo</i> binding) (C). 293T cells were transfected with expression plasmids for FLAG-BNIP-H full length, FLAG-BNIP-H aa 1–287, FLAG-BNIP-H Δ aa 189–287 or left untransfected. Lysates were subjected to immunoprecipitation with anti-FLAG antibody conjugated to agarose beads, and precipitates were washed thoroughly with RIPA buffer and RIPA buffer supplemented with 0.1–0.5% sodium dodecyl sulfate or 500 mM NaCl as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002686#s4" target="_blank">Material and Methods</a>”. Precipitates were resuspended in lysis buffer and incubated with recombinant Pin1 expressed and purified from <i>E.coli</i>. After sedimentation and washing, precipitates were analyzed by western-blotting with anti-Pin1 antibody (lanes 6–9). Lane 1 shows 10% of the input for recombinant Pin1. Lanes 2–5 demonstrate that the purified FLAG-BNIP-H constructs were devoid of endogenous Pin1 from 293T cells. The amido black-stained membrane shows equal input for the FLAG-BNIP-H constructs.