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. 2004 May 5;24(18):4401–4411. doi: 10.1523/JNEUROSCI.0348-04.2004

Figure 3.

Figure 3.

Coimmunoprecipitation of BDNFVal and BDNFMet heterodimers. A, To detect different degrees of heterodimerizatiom of two epitope-tagged forms of wild-type BDNF, COS-7 cells were transfected with two wild-type BDNF constructs bearing different C-terminal epitopes (HA or FLAG). The amount of cotransfected FLAG-BDNF and HA-BDNF was varied while maintaining a constant amount of total DNA (12 μg) transfected. Cell lysates were prepared as described in Materials and Methods, and the first immunoprecipitations were performed with HA antibodies (IP#1 indicates HA). A second immunoprecipitation was subsequently performed on the remaining lysates with FLAG antibodies (IP#2 indicates FLAG), and immunoprecipitated samples were analyzed by Western blot. In addition, initial lysates (20 μg of total protein) were analyzed by Western blot for levels of transfected FLAG-BDNF and HA-BDNF. B, To detect different degrees of heterodimerizatiom of epitope-tagged forms of wild-type and variant BDNF, COS-7 cells were transfected with combinations of BDNFVal and BDNFMet constructs bearing different C-terminal epitopes (HA-BDNFVal plus FLAG-BDNFVal, HA-BDNFMet plus FLAG-BDNFMet, HA-BDNFVal plus FLAG-BDNFMet, or FLAG-BDNFVal plus HA-BDNFMet). A constant amount (6 μg) of each epitope-tagged BDNF species was transfected to maintain a constant amount of total DNA (12 μg) transfected. Cell lysates were prepared as described in Materials and Methods, and sequential immunoprecipitations were performed (IP#1 indicates HA; IP#2 indicates FLAG), as described in A, and analyzed by Western blot. In addition, initial lysates (20 μg of total protein) were analyzed by Western blot for levels of transfected BDNFVal and BDNFMet. C, Quantitation of Western blots in A as a proportion of FLAG-BDNF immunoreactivity found in IP#1 over the total FLAG immunoreactivity obtained from both immunoprecipitation steps (IP#1 and IP#2, Blot: FLAG). Mean ± SEM proportions were determined from analysis of three independent experiments. D, Quantitation of Western blots in B as a proportion of FLAG-BDNF immunoreactivity found in IP#1 over the total FLAG immunoreactivity obtained from both immunoprecipitation steps (IP#1 and IP#2, Blot: FLAG). Mean ± SEM proportions were determined from analysis of three independent experiments.