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. Author manuscript; available in PMC: 2022 May 12.
Published in final edited form as: Dev Cell. 2021 Mar 15;56(6):811–825.e6. doi: 10.1016/j.devcel.2021.02.022

Figure 4. Rheb interacts with PDH phosphatases (PDPs).

Figure 4.

(A) GST pull-down demonstrating that purified GST-Rheb binds endogenous PDP1 in the brain (A, top panel) and PDP2 in the liver (A, middle panel).

(B) Images showing PLA signals (indicated by arrow heads) in HeLa cells transfected with GFP-Rheb using GFP and PDP1 antibody.

(C) GST pull-down demonstrating that GST-Rheb binds HA-tagged PDP2 transiently expressed in HEK293T cells. Note that GST-Rheb does not pull down HA-PP2Cα1 transiently expressed in HEK293T cells.

(D) Coimmunoprecipitation of myc-Rheb and HA-PDP2 in HEK293T cells demonstrating the interaction of Rheb and PDP2.

(E) In vitro reconstitution of direct binding between bacterially expressed purified GST-Rheb and recombinant His-PDP2 purified from HEK293T cells.

(F) In vitro reconstitution of direct binding between bacterially expressed recombinant His-Rheb and GST-PDP2.

(G) GST pull-down demonstrating that purified GST-PDP2 binds myc-tagged Rheb (wild type) and D60K mutant transiently expressed in HEK293T cells.

(H) GST pull-down demonstrating that purified HA-GST-Rheb loaded with GTPγS, GDP, or nucleotide-free (free) binds HA-tagged PDP2 transiently expressed in HEK293T cells. See also Figure S4.