Figure 4. AKAP121 Facilitates the Phosphorylation of Drp1 at Mitochondria Microenvironment.

(A and B) Siah1a/2^DKO and Siah1a/2^DKO MEFs stably expressing scrambled or shRNA against AKAP121 (A) or Siah1a/2^WT and Siah1a/2^DKO MEFs (B) were stimulated with forskolin (10 µM) and cyclosporine (10 µM) for 30 min. Cells were fractionated as described in the Experimental Procedures.

(C) HEK293T cells were transfected with AKAP121-Flag. Lysates from each fraction were analyzed with indicated antibodies.

(D) HEK293T cells transfected with indicates constructs. Lysates were immunoprecipitated with Flag (AKAP121) and immuoblotted with HA (Drp1 or Drp1Δ256 [lacking GTPase domain]).

(E) HEK293T cells transfected with Drp1-myc and indicated constructs of AKAP121, N350 (1–350), N564 (1–564), C351 (351–857), and C537 (537–857). Lysates were immunoprecipitated and blotted with indicated antibodies.

(F) Cells were transfected with indicated constructs and treated with forskolin (10 µM)/cyclosporineA (10 µM) for 30 min.

(G) Cells were transfected with AKAP121 and Drp1 wild-type (W), phosphormutant S637A(S/A), and phosphomimic mutant S637D (S/D).

(F and G) Intensity of immunoprecipitated Drp1 bands was quantified by normalizing with corresponding input for Drp1. The values in the control groups were set to 1.