DUF581-9 interferes with KIN10/GRIK2 interaction in vivo and in vitro leading to diminished KIN10 T175 T-loop phosphorylation in vivo. A) Validation of KIN10 and GRIK2 interaction after KIN10 myc:IP (immunoprecipitation). Furthermore, KIN10 T-loop phosphorylation is weakened by DUF581-9 but not by KIN10-binding inefficient DUF581-9_C47S protein. Proteins were transiently expressed in leaves of N. benthamiana using Agrobacterium infiltration. Samples were harvested 48 h post infiltration. Crude extract mirrors input signal of KIN10 (anti-myc), GRIK2 or KINß2 (anti-HA), and DUF581-9/DUF581-9_C47S (anti-mCherry) protein. For KIN10 T-loop phosphorylation, immunoblot analysis was performed with anti-P T172 AMPK antibody. Densitometric analysis of anti-P T172 AMPK signal confirms the weaker KIN10 T-loop phosphorylation only in the presence of DUF581-9 protein. Pulldown of KIN10 protein was performed by anti-myc affinity matrix followed by immunoblot analysis using anti-myc, anti-HA, or anti-mCherry antibodies. Densitometric analysis of KIN10 Venus^N173 and GRIK2-Venus^C155 association confirms the weaker protein interaction of KIN10 and GRIK2 in the presence of DUF581-9. The homomerization of KIN10-Venus^N173 and KINß2-Venus^C155 was used as control and is not affected by DUF581-9 mCherry and DUF581-9_C47S mCherry. B) Proteins were mixed as indicated, and KIN10-GST (full length) was pulled down from the mixture. Recombinant proteins were detected before (Input) and after (IP:GST) by immunoblotting using anti-MBP or anti-GST antibodies. Red box highlights GRIK2 MBP protein abundance after KIN10 IP:GST. The experiment was carried out at least twice with similar results. Asterisks indicate an unspecific protein band present in MBP empty vector control.