Fig. 6: Ternary complex formation of KL, MDM2, and SHANK1 in vitro and in vivo.

A Co-immunoprecipitation of MDM2 with KL. Lysates from A549 cells transfected with MDM2-HA, KL-Flag, or sh-SHANK1 constructs were detected by immunoprecipitation and immunoblotting analysis. B Lysates from A549 cells transfected with SHANK1-myc or sh-SHANK1 vector were detected by immunoblotting analysis to show the protein expression of MDM2. C, D Endogenous MDM2 associates with SHANK1 and KL. A549 cell lysates were subjected to immunoprecipitation with mouse anti-MDM2 or Rabbit anti-KL antibodies, and then the protein complex was eluted. MDM2, KL, and SHANK1 were detected by immunoblotting. E Lysates from A549 cells co-transfected with Myc-SHANK1/or si-SHANK1. MDM2-HA and Flag-KL constructs were immunoprecipitated with anti-HA antibodies. Immunoblotting analysis was then performed to detect immunoprecipitated proteins. F Lysates from A549 cells co-transfected with Myc-SHANK1, MDM2-HA/or si-MDM2, and Flag-KL constructs were immunoprecipitated with anti-Myc antibodies. Next, immunoblotting analysis was performed to detect immunoprecipitated proteins. G Lysates from A549 cells transfected with si-SHANK1 were collected, and then immunoblotting analysis was performed by KL antibody to detect immunoprecipitated proteins. H Lysates from H1299 cells transfected with si-SHANK1 were collected, and then immunoblotting analysis was performed by KL antibody to detect immunoprecipitated proteins. I Lysates from A549 cells co-transfected with Myc-SHANK1/or si-SHANK1, MDM2-HA, and Flag-p53 constructs were immunoprecipitated with anti-HA antibodies. J Lysates from H1299 cells co-transfected with Myc-SHANK1/or si-SHANK1, MDM2-HA, and Flag-p53 constructs were immunoprecipitated with anti-HA antibodies. K A549 cells were transfected with vector or SHANK1-myc constructs and lysed by TNE buffer, followed by immunoblotting with anti-p53 and p21 antibodies.