1. Volume 18, Issue 13, Pages 3069-3077 (March 2017)
Identification of a Tissue-Restricted Isoform of SIRT1 Defines a Regulatory Domain that Encodes Specificity 
Shaunak Deota, Tandrika Chattopadhyay, Deepti Ramachandran, Eric Armstrong, Beatriz Camacho, Babukrishna Maniyadath, Amit Fulzele, Anne Gonzalez-de-Peredo, John M. Denu, Ullas Kolthur-Seetharam 
Cell Reports 
Volume 18, Issue 13, Pages (March 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 18, 3069-3077DOI: (10.1016/j.celrep.2017.03.012)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1 Identification of a Novel Isoform of SIRT1 Lacking E2 Domain
(A) Immunoblot of SIRT1 using α-SIRT1 (N: 1-131) antibody.
(B) Schematic of full-length mouse SIRT1 protein indicating the antigenic regions targeted by the different antibodies. MATG marks the ATG at the 21st codon.
(C) Immunoblot of SIRT1 immunoprecipitated from mouse testis nuclear lysate with the indicated antibodies. B1 and B2 represent the slower and faster migrating SIRT1 bands, respectively.
(D) Immunoblot of SIRT1 from lysates treated with or without calf intestinal phosphatase (CIP), using pAKT/AKT as a positive control.
(E) Immunoblot of SIRT1 from lysates treated with or without calf intestinal phosphatase (CIP) shows both the isoforms of SIRT1 in testis.
(F) Immunoblot of SIRT1 from wild-type (WT) (Exon-4lox/lox) and SIRT1 KO (Exon-4Δ/Δ) mice.
(G) Peptide fingerprinting of B1 and B2 bands of SIRT1 immunoprecipitated from mice testes nuclear lysate and mapping of exon-specific peptides.
(H) Schematic of primer locations.
(I) RT-PCR of Sirt1-FL/-ΔE2 using indicated primer pairs from mRNA. L, liver; T, testis.
See also Figure S1.
Cell Reports  , DOI: ( /j.celrep )
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4. Figure 2
Expression Pattern, Catalytic Properties, and Dimerization Potential of SIRT1-FL and SIRT1-ΔE2
(A) RT-PCR of Sirt1-FL/-ΔE2 in mice tissues using F1 and R2 primers (refer Figure 1H). PCR products from the intestine were run separately.
(B) qPCR of Sirt1-FL from indicated tissues.
(C) Ratio of Sirt1-FL/-ΔE2 mRNA from indicated tissues.
(D and E) qPCR of Sirt1-FL/-ΔE2 mRNA from young/old testis (D) and hippocampus and cortex (E).
(F) Immunoblot of SIRT1 isoforms from young and old mice.
(G) Nucleo-Cytoplasmic distribution of SIRT1-FL/-ΔE2.
(H) Immunoblot of SIRT1 isoforms from chromatin and nucleoplasmic fractions.
(I) Immunoblot of SIRT1-FL or SIRT1-ΔE2 in immunoprecipitates from HEK293T expressing Myc-SIRT1-ΔE2 with either FLAG-SIRT1-FL or FLAG-SIRT1-ΔE2.
(J and K) Substrate saturation curves of (J) bacterially expressed mSIRT1-ΔE2 and hSIRT1-FL or (K) mammalian expressed mSIRT1-ΔE2 and mSIRT1-FL.
(L) NAD+ saturation curves of mSIRT1-ΔE2 and mSIRT1-FL. Lines represent fits to the Michaelis-Menten equation, v = Vmax∗[S]/([S] + KM).
(M) Domain structure of SIRT1. Error bars reflect SD (J–L) or SEM (B–E) from two to three replicates.
See also Figure S2.
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5. Figure 3
E2 in SIRT1-FL Is Required for Interaction, Deacetylation, and Inhibition of p53
(A and B) Immunoblot of Ac-p53 (K382) in HEK293T expressing FLAG-SIRT1-FL/-ΔE2 (A) in basal conditions and (B) following treatment with 200 μM H2O2 for 2 hr.
(C) p53 reporter luciferase assay in HEK293T expressing p53 and FLAG-SIRT1-FL/-ΔE2.
(D–F) Immunoblot of Myc or FLAG-M2 immunoprecipitates from HEK293T expressing (D and E) Myc-DBC1 with FLAG-SIRT1-FL/-ΔE2, for interaction with DBC1 (LE/HE, low/high exposures) and (F) FLAG-SIRT1-FL or Myc-SIRT1-ΔE2 for interaction with p53.
(G) In vitro pull-down from HEK293T lysates using GST, GST-E2, GST-E1-E2-E3, or GST-E1-E3 and immunoblotting for endogenous p53. Error bars indicate SEM (n = 3 of two independent experiments).
See also Figure S3.
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6. Figure 4
Differential Binding Specificities of SIRT1 Interactors Encoded by E2 Affect Insulin Signaling, Gene Expression, and FAO
(A) Immunoblot of FLAG-M2 or Myc immunoprecipitates from HEK293T expressing either FLAG SIRT1-FL or Myc SIRT1-ΔE2 for interaction with PGC1α, PPARα, FOXO1, and FOXO3a.
(B) In vitro pull-down from HEK293T, C2C12, testis, or liver lysates using GST, GST-E2, GST-E1-E2-E3, or GST-E1-E3 and immunoblotting for endogenous PGC1α. Different exposures of input and pull-down from the same blot are shown separately.
(C) PPARα reporter luciferase assay in HEK293T expressing PPARα and FLAG-SIRT1-FL/-ΔE2.
(D) Immunoblot of FLAG-M2 immunoprecipitates from HEK293T expressing FLAG-SIRT1-FL/-ΔE2 for interaction with AKT and CREB.
(E) Immunoblot of pAKT/AKT in HeLa cells expressing FLAG-SIRT1-FL/-ΔE2 in response to insulin stimulation (100 nM/15 min).
(F) FLAG-M2 immunoprecipitates from HEK293T expressing HA-TIF1β and FLAG-SIRT1-FL/-ΔE2 for interaction with HA-TIF1β.
(G) qPCR of indicated downstream genes from HEK293 cells expressing FLAG-SIRT1-FL/-ΔE2.
(H) Palmitate-induced oxygen consumption rate (OCR-FAO) in HepG2 cells expressing FLAG-SIRT1-FL/-ΔE2.
Error bars indicate SEM (n = 3 of two independent experiments).
See also Figure S3.
Cell Reports  , DOI: ( /j.celrep )
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