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. Author manuscript; available in PMC: 2014 Aug 15.
Published in final edited form as: Exp Cell Res. 2013 Jun 25;319(14):2275–2281. doi: 10.1016/j.yexcr.2013.06.010

Figure 3. Interaction of TTC30B with multiple IFT proteins in a cell-free, in vitro system.

Figure 3

Coding sequences for tagged proteins were prepared by PCR and fused to a promoter for bacteriophage SP6 polymerase. The promoter-tagged protein DNA was used to direct protein synthesis in a cell-free coupled transcription-translation system from wheat germ. The in vitro reactions contained either coding sequences for AU1-tagged TTC30B or FLAG-tagged IFT protein as indicated. (A) To analyze protein expression, aliquots of the in vitro reactions were resolved by denaturing gel electrophoresis and immunoblotting with antibody to FLAG to detect the input expression of IFT proteins or with AU1 antibody to detect input expression of TTC30B. non-specific band was detected in all lanes and serves as a loading control. (B) To detect interaction of TTC30B with IFT proteins, FLAG-tagged IFT proteins were isolated by immunoprecipitation with FLAG antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with AU1 antibody to detect co-immunoprecipitate, AU1-tagged-TTC30B.