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. 2012 Sep 3;198(5):895–911. doi: 10.1083/jcb.201201139

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© 2012 Xu et al.

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Figure 3. — Physical interaction between mammalian FATP1 and DGAT2. (A) ACS-22–2×HA coimmunoprecipitated with FLAG-DGAT-2 from HEK293T cell extracts. 6% input of each sample was loaded. (B) Schematic representation of mouse FATP1 and DGAT2 proteins. Minimal regions that are required for FATP1–DGAT2 interaction are shaded in dark gray. (C) HA-FATP1 preferentially coimmunoprecipitated with FLAG-DGAT2 from HeLa cell extracts. FLAG-DGAT1 was expressed at very high level, yet showed weaker association with HA-FATP1. HA-FATP1 also associated with FLAG-FATP1 but not FLAG-Venus. 5% input of each sample was loaded for HA-FATP1, and 10% input of each sample was loaded for FLAG-tagged proteins. The input of FLAG-DGAT2 (lane 2) was not detectable because of its low level of expression. The asterisk indicates immunoglobulin light chains. (D) FLAG-DGAT2 (black arrowhead) associated with wild-type and mutant HA-FATP1 with similar affinity in HEK293T cell extracts. FLAG-Venus was indicated by the gray arrowhead. 5% input of each sample was loaded for HA-FATP1, and 10% input of each sample was loaded for FLAG-tagged proteins. The FLAG-DGAT2 signal was obscured by background signals in lanes 2–5. The asterisks indicate immunoglobulin light chains and a nonspecific protein larger than DGAT2 in lanes 6–10. (E) Coimmunoprecipitation of in vitro translated HA-FATP1 with FLAG-DGAT2 (black arrowhead) but not FLAG-Venus (gray arrowhead). 3% input of each sample was loaded for HA-FATP1, and 13% input of each sample was loaded for FLAG-tagged proteins. The asterisk indicates a nonspecific protein. (F) Coimmunoprecipitation of in vitro translated HA-FATP1 with deletion mutants of FLAG-GST-DGAT2. 1.4% input of each sample was loaded for HA-FATP1, and 28% input of each sample was loaded for FLAG-tagged proteins. The asterisk indicates nonspecific proteins. The black arrow indicates FLAG-GST-DGAT2(116–228) that partially overlaps with a nonspecific band. (G) Coimmunoprecipitation of in vitro translated HA-FATP1 deletion mutants with FLAG-DGAT2 (black arrowhead). 1.4% input of each sample was loaded for HA-FATP1, and 56% input of each sample was loaded for FLAG-tagged proteins. The asterisk indicates a nonspecific protein. IB, immunoblotting; IP, immunoprecipitation.