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Results: 4

1.
Figure 2

Figure 2. Non-phosphorylation of Thr522 results in formation of SIRT1 protein aggregates in vitro.. From: The NAD+-dependent protein deacetylase activity of SIRT1 is regulated by its oligomeric status.

(A) The SIRT1 TE mutant proteins displays smaller size than nonphosphorylated SIRT1 proteins (WT and TA) in solution. Purified recombinant SIRT1 proteins were incubated overnight at 4, 30, and 45°C respectively, and the solution size of these proteins were determined by dynamic light scattering as described in the Methods. (B) The nonphosphorylated SIRT1 proteins (WT and TA) are aggregation-prone, whereas the TE mutant proteins are primarily monomers in solution. Purified recombinant SIRT1 proteins were kept overnight at 4°C and then analyzed at room temperature by sizing-column coupled multi-angle light scattering as described in the Methods and the molecular weight is calculated across the distribution. (C) Representative negatively stained EM images of samples used in B. Arrow heads mark SIRT1 oligomers and aggregates in the WT and TA samples.

Xiumei Guo, et al. Sci Rep. 2012;2:640.
2.
Figure 1

Figure 1. Phosphorylation of Thr522 activates SIRT1 in vitro.. From: The NAD+-dependent protein deacetylase activity of SIRT1 is regulated by its oligomeric status.

(A) Representative in vitro enzymatic analysis of 6xHis tagged WT, TA, and TE SIRT1 proteins. 0.02 μg of purified recombinant WT, TA, or TE SIRT1 proteins were incubated with 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, and 3.0 μg of in vitro acetylated GST-p53 fusion proteins in the presence of 3 mM of NAD+ and 200 nM of TSA, and incubated at 37°C for 30 min. The acetylated GST-p53 levels were analyzed as described in the Methods. (B) Quantification of three independent in vitro enzymatic experiments in A. The total and acetylated GST-p53 levels were quantified as described in the Methods. (C) Representative in vitro enzymatic analysis of 6xHis tagged WT SIRT1 (WT) and DYRK1A-phosphorylated WT SIRT1 (p-WT) proteins. The assays were preformed as described in (A) and the rate of deacetylated p53 levels were analyzed as described in the Methods. (D) Quantification of three independent in vitro enzymatic experiments as in C. The total and acetylated GST-p53 levels were quantified as described in the Methods. (*, p<0.05).

Xiumei Guo, et al. Sci Rep. 2012;2:640.
3.
Figure 3

Figure 3. Phosphorylation of Thr522 activates SIRT1 through preventing the formation of less-active SIRT1 aggregates in cells.. From: The NAD+-dependent protein deacetylase activity of SIRT1 is regulated by its oligomeric status.

(A) Abnormal accumulation of the SIRT1 TA mutant proteins near the peripheral of nucleus in cells. HEK293T T1RNAi cells were transfected with constructs expressing indicated SIRT1 proteins. The distribution of these proteins was analyzed by Immuno-fluorescent staining and confocal microscopy imaging as described in the Methods. (B) The distribution of SIRT1 WT, TA, and TE proteins in 100 nuclei from experiments in A was analyzed along the nuclear diameter as described in the Methods. (C–D) The SIRT1 TA mutant protein is prone to aggregation upon heat shock in cells. HEK293T T1RNAi cells transfected with constructs expressing indicated SIRT1 proteins were heat shocked at 45°C for 15 min and cooled down for 15 min on ice. Cells were then crosslinked with 0.1% glutaraldehyde/PBS on ice for indicated time (C) or 30 min (D) and analyzed by SDS-PAGE and immunoblotting (C) or filter dot-blotting (D) as described in the Methods.

Xiumei Guo, et al. Sci Rep. 2012;2:640.
4.
Figure 4

Figure 4. Phosphorylation of Thr522 prevents formation of SIRT1 aggregates and promotes cell survival in response to heat shock in knockin MEFs.. From: The NAD+-dependent protein deacetylase activity of SIRT1 is regulated by its oligomeric status.

(A) The phosphorylation status of SIRT1 does not affect the degradation of SIRT1 proteins in MEFs upon heat shock treatment. The MEFs expressing endogenous WT SIRT1 proteins, or knocked-in TA (TAKI) or TE (TEKI) mutant proteins were incubated with 10 μg/ml of cycloheximide (CHX) upon heat shock at 45°C, the levels of SIRT1 were analyzed by immuno-blotting at indicated time points after treatment. (B) The SIRT1 TE mutant proteins are protected from heat-shock induced aggregation in MEFs. Indicated MEFs were heat shocked at 45°C for 30 min and analyzed by crosslinking as described in the Methods. (C) The SIRT1 TE knock-in MEFs are more resistant to heat shock. Indicated MEFs were treated at 45°C for 1 h, and then recover at 37°C for 24 h. Cell viability was determined as described in the Methods. (D) Phosphorylation of Thr522 activates SIRT1 through preventing the formation of less-active SIRT1 oligomers. (*, p<0.05).

Xiumei Guo, et al. Sci Rep. 2012;2:640.

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