Fig. 3. Overlay analysis of binding interactions between CaP and supervillin/archvillin fragments. (A, left) Naphthol blue black (NBB) stain of total protein on blot before incubation with overlay solution. (A, right) Immunostain with anti-CaP antibody after processing with overlay buffer containing 1.25 µg ml^-1 CaP. Purified proteins and their corresponding overlay signals in lanes are indicated with arrowheads. All three proteins were loaded at 0.2 µg per lane. (B) Quantitative analysis of results obtained in (A). The chart shows the ratio of densitometry obtained from the overlay assay to that from the NBB-stained blot. The results are from n=7 (GST-c-BSV), n=6 (GST-c-SmAV) and n=3 (GST) samples in each case. (C, left) NBB stain of total protein on blot before incubation with overlay solution. (C, right) Immunostain with anti-GST antibody after processing with overlay buffer containing 1.25 µg ml^-1 GST-c-SmAV or GST only as indicated. Purified proteins and their corresponding overlay signals in lanes are indicated with arrowheads. Both recombinant CaP and recombinant calmodulin were at 3 µg per lane. (D) Quantitative analysis of results obtained in (C). The chart shows the ratio of densitometry obtained from the overlay assay to that from the NBB-stained blot. The results are from n=5 (CaP) and n=3 (CaM) samples for the GST-c-SmAV overlay, and n=3 (CaP) and n=3 (CaM) samples for the GST overlay. (E) Overlay assay of GST-c-SmAV with chymotrypsin-digested fragments of CaP. (left) NBB stain of blot before incubation with overlay solution. (right) Immunostain with anti-GST antibody after processing with overlay buffer containing 1.25 µg ml^-1 GST-c-SmAV. Undigested (full-length) and fragments of CaP are indicated with arrowheads. (F) Quantitative analysis of results obtained in (E). The chart shows the ratio of densitometry obtained from the overlay assay to that from the NBB-stained blot. The results are from n=5 samples.