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. 2013 Mar 14;8(3):e58814. doi: 10.1371/journal.pone.0058814

Figure 4. Bassoon interacts with 14-3-3 in living cells. (A).

Figure 4

GFP-Bsn28 (wt) wild-type or its mutant GFP-Bsn28S2845A were expressed in HEK293T cells and cell lysates were used for immunoprecipitations with α-GFP antibody. Successful expression of GFP-tagged proteins was shown in cell lysates (input) and bound fraction (IP) using a-GFP antibody. The endogenously expressed 14-3-3 proteins were co-precipitated with GFP-Bsn28 but not with its mutant Bsn28S2845A as demonstrated by detection with α-pan 14-3-3 and α-14-3-3η antibody. Note two bands detected in the bound fractions using α-pan 14-3-3 antibody suggesting immunoprecipitation of multiple 14-3-3 isoforms. The bars and number on left side of blots show the sizes and positions of molecular weight markers. Images shown are representative for results obtained in two independent experiments. (B, C) mRFP-14-3-3η is localized to clusters formed by GFP-Bsn (arrows in B) but not to clusters formed by the corresponding 14-3-3 binding mutant GFP-BsnS2845A (arrows in C) in transfected primary hippocampal neurons. Scale bar is 20 µm.