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. 2005 May 10;388(Pt 1):195–204. doi: 10.1042/BJ20041797

Figure 2. Levels and distribution of STNB, DAP-160 and dynamin in stoned mutants.

Figure 2

(A) Equivalent volumes (see the Experimental section) of P1, P2, P3 and S3 fractions from homogenates of heads of w stnC and w1118 control flies, were subjected to SDS/PAGE (7% gel), Western blotted and probed with anti-STNB antibodies. The expected >220 kDa protein is absent from all but the P3 fraction of w stnC. A smaller 85 kDa protein can be seen in the P2 and S3 from w stnC flies. The same fractions were also probed with anti-DAP-160, anti-dynamin (DYN) and anti-STNA antibodies. A very obvious reduction in the levels of DAP-160 and dynamin is observed, but there is little effect on STNA levels. (B) For each lane, six heads from w stnC and w1118 control flies were homogenized directly into SDS/PAGE sample buffer, boiled and centrifuged (10000 g for 5 min) to remove particulate material, and loaded on an SDS/7% polyacrylamide gel. The resulting blot was then probed with three preparations of anti-STNB antibodies, those described in the present report and raised against the N-terminal fragment of STNB, those raised against the C-terminal region of the protein [10] or raised against a mixture of an N-terminal and C-terminal peptide [15]. (C) A blot the same as that for (B) was prepared and overlayed with either GST alone or a GST–DAP-160(EH2) fusion protein. The overlay blots were developed with anti-GST polyclonal antibodies. This shows that both N-terminal anti-STNB antibodies and the GST–DAP-160(EH2) overlay method both identify a 85 kDa truncated protein in the w, stnC extracts that is absent when using the C-terminal-specific antibodies. (D) P1, P2, P3 and S3 fractions from homogenates of heads from w stnts and w1118 control flies were run on SDS/PAGE (7% gel) as in (A), Western blotted and probed with anti-STNB, anti-DAP-160 and anti-dynamin antibodies. While there is a reduction in the levels of STNB in w stnts heads, both DAP-160 and dynamin are at normal levels and there is little difference in their distribution. (E) Heads (five) from w1118, EP2543, w stnC:EP2543 and w stnC were treated as for (A). The resulting Western blot was then probed with anti-DAP-160 antibodies. All of the mutant flies showed the same level of DAP-160, which corresponds to approx. 50% of the wild-type levels.