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. 2021 Sep 27;24(11):103177. doi: 10.1016/j.isci.2021.103177

Figure 4.

Figure 4

Acetylated-FKBP12 associates with acety-Rheb contributing to mTORC1 kinase activation

(A) HEK293T cells transfected with FLAG-FKBP12 and Myc-Rheb followed by AA treatment for indicated times. Anti-Flag IP was analyzed for FKBP12 and Rheb interaction in Western blot.

(B) In fibroblasts, 20% FBS treatment for indicated time. WCLs prepared for Rheb IP followed by pan acetyl-K blotting.

(C) Mass spectra of acetyl-K8, acetyl-K169 and acetyl-K120 peptides of Rheb recovered from trypsin digests.

(D) Alignment of double-positive “RK” motif of Rheb of different species with “RK” motifs of histone H3.

(E) HA-FKBP12 and Myc-Rheb were cotransfected in HEK293T cells. NAM or TSA treatment for 2 h was tested on FKBP12 and Rheb interaction.

(F) Two reporter system (Rheb-N-Luc and FKBP12-C-Luc of WT, K48R or K48Q) were cotransfected and treated with rapamycin treatment for 6 h followed by the luciferase reporter activity analysis. Data are represented as mean ± SD; ∗∗∗∗, p < 0.0001, one-way ANOVA.

(G) Flag-Rheb WT or variant (K8R, K8Q) or EV was cotransfected with HA-FKBP12 in HEK293T cells. Anti-HA IP was analyzed for FKBP12 and Flag-Rheb interaction in Western blot with anti-Flag. WCLs were analyzed for S6K1 phosphorylation with anti-S6K1 pT389.