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

Figure 2.

Figure 2

SIRT2 deacetylates FKBP12

(A) FKBP12 deacetylation induction by rapamycin in mouse fibroblasts for indicated times and blotted with indicated antibodies.

(B) In HEK293T cells, HA-FKBP12 and FLAG-CBP were cotransfected followed by NAM, or TSA treatment as indicated. Anti-HA IP was analyzed for FKBP12 acetylation with pan acetyl-K antibody in Western blot. FKBP12-acK band intensities were measured by ImageJ software and normalized to FKBP12 (right panel). Data are represented as mean ± SD; ∗∗∗∗, p < 0.0001, one-way ANOVA.

(C) HeLa cells were treated with indicated doses of RC32 for 12 h. WCLs were analyzed for SIRT2 or FKBP12 degradation with indicated antibodies.

(D) Rapamycin binding affinity Kds were obtained from ITC analysis of indicated proteins purified from bacteria.

(E) Purified SIRT2, HDAC1, and FRB proteins were incubated with rapamycin for MST analysis.

(F) Naïve PAGE detection of rapamycin ternary complex formation with FKBP12 (left) or with SIRT2 (right). GST-FKBP12, GST-SIRT2 and GST-HDAC1 were purified from bacteria and incubated with rapamycin prior to loading onto the naive PAGE and blotted with anti-GST.