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. Author manuscript; available in PMC: 2021 Mar 28.
Published in final edited form as: Nat Cell Biol. 2020 Sep 28;22(10):1170–1179. doi: 10.1038/s41556-020-00579-5

Figure 4. SIRT1 interacts with LC3 through a LIR motif.

Figure 4.

a, SIRT1 peptide array showing potential interaction regions with LC3. 20-mer peptides covering full-length SIRT1 with a moving window of 3 residues were synthesized and incubated on a cellulose membrane. The array was probed with GST-LC3B. Eight potential regions for LC3 binding were boxed and numbered. b, IP of HEK293T cells expressing HA-tagged SIRT1 and Flag-tagged LC3, with addition of DMSO or 500 μM synthetic SIRT1 peptides as indicated; n = 3 independent experiments. c, IP of HEK293T cells expressing HA-tagged SIRT1 WT, W221A/V224A (WV), F474A/D475A/V476A or Y497A/L500A substitutions; n = 2 independent experiments. d, IP of HEK293T cells expressing Flag-tagged LC3 and HA-tagged SIRT1 WT, WV or E214A/D216A/D217A (EDD) mutants; n = 2 independent experiments. e, Scheme of SIRT1 showing the location of the 205–233 peptide. Potential amino acid residues involved in LC3 binding are labeled in red, including the core LIR motif WQIV. Location of 205–233 peptide is labeled in blue; locations of other peptides tested are labeled in grey. f, IP of HEK293T cells expressing HA-tagged SIRT1 and Flag-tagged LC3, with addition of DMSO or 500 μM synthetic SIRT1 peptides as indicated; n = 2 independent experiments. g, IMR90 cells stably expressing HA-tagged SIRT1 I347A or WV+I347A mutants were infected with HRasV12 retrovirus, selected by antibiotics and harvested at indicated days for western blotting; n = 2 independent experiments. h-i, Senescent IMR90 cells expressing mCherry-GFP-SIRT1 I347A or WV+I347A mutants were analyzed. Senescence was initiated by etoposide treatment for indicated days. h, Cells at day 7 after etoposide treatment were imaged by confocal microscopy. Scale bar: 5 μm. i, Percentages of cells with cytoplasmic SIRT1 puncta were quantified. Data are mean ± s.d.; more than 500 cells were counted; each data point (n) represents cells in 10 random fields, n = 5 for all conditions; two-way ANOVA with Sidak’s multiple comparisons test. Statistical information and unprocessed blots are provided as source data.