Anti-SIRT6 antibody

• WB

Unknown

Western blot - Anti-SIRT6 antibody (AB62739)

Lane 1:

Western blot - Anti-SIRT6 antibody (ab62739) at 0.5 µg/mL

Lane 2:

Western blot - Anti-SIRT6 antibody (ab62739) at 1 µg/mL

Lanes 3 - 4:

Western blot - Anti-SIRT6 antibody (ab62739) at 2 µg/mL

Lanes 1 - 3:

Mouse 3T3 whole cell extract

Lane 4:

Mouse 3T3 whole cell extract with immunizing peptide

Secondary

All lanes:

Goat Anti-Rabbit IgG,

Predicted band size: 39 kDa

Observed band size: 37 kDa

true

• ICC

Supplier Data

Immunocytochemistry - Anti-SIRT6 antibody (AB62739)

Immunocytochemistry/ Immunofluorescence analysis of HeLa cells labeling SIRT6 with ab62739. Cells were fixed and permeabilized with 4% paraformaldehyde followed by 0.5% Triton^™ X-100. Fixed cells were stained with 2.5 μg/mL Anti-SIRT6 antibody - ChIP Grade (ab62739). The antibody was developed using Goat Anti-Rabbit IgG, Cy3^™ conjugate.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-SIRT6 antibody (AB62739)

Immunocytochemistry/ Immunofluorescence analysis of mouse primary hepatocytes labeling SIRT6 with ab62739 at 1/200 dilution. The cells were fixed with paraformaldehyde, followed by blocking with 3% BSA for 2 hours at 20°C. A polyclonal goat anti-rabbit IgG Alexa Fluor^® 488 secondary antibody was used at 1/10000 dilution.

This image is courtesy of an anonymous abreview.

• WB

CiteAb

Western blot - Anti-SIRT6 antibody (AB62739)

Western Blotting using Anti-SIRT6 antibody, ab62739. Publication image from Santos-Barriopedro, I. et al., 2018, Nat Commun, 29317652. Legend direct from paper.

SirT6 regulates nuclear levels of SKP2 through deacetylation. a Analysis of the effect of SirT6 on the SKP2 levels. 293F and HeLa cells were co-transfected with the indicated combinations of FLAG-SKP2 and SirT6-HA. Tubulin was included as loading control. b Levels of acetylation in FLAG-SKP2 purified from 293F cells downregulated in SirT6 (shScramble vs shSirT6, left) or overexpressing SirT6-HA in 293F cells expressing shRNA SirT6 (right). SKP2 was purified with FLAG resin and analyzed by western-blot with anti-acetyl-lysine antibodies. c Posttranslational modifications in SKP2 in the presence or absence of SirT6. Upper panel : Summary of the procedure. Lower panel : Summary of the identified PTMs in SKP2 in the indicated conditions. Additional data is shown in Supplementary Data Set 2. d Left, ESI-MS spectrum of acetylated peptide (aa 72–83, 72K.SK73acGSDK77acDFVIVR.R.83-C) from SKP2 − / + SirT6 of a representive experiment of two replicas. Signal was detected at m/z 464.92 (charge state 4). Middle and right, ESI-MS spectrum of phosphorylated peptides (aa 72–83, 72K.S72phKGS75phDKDFVIVR.R.83-C, 72K.KGS75phDKDFVIVR.R.83-C) from SKP2 − / + SirT6. Signal appeared at m/z 477.61 and 477.61, respectively). e Analysis of the ability of SirT6-HA to upregulate the levels of nuclear FLAG-tagged SKP2, as either WT or tetramutated in K68R/K71R/K73R/K77R. Western blotting of nuclear extracts from 293F cells previously transfected with the indicated constructs. f Quantification of n = 5 experiments as the one showed in e. SKP2 levels were normalized with histone H3. All the values were represented relative to the normalized levels of WT SKP2 in the absence of SirT6 (T-test; s.e.m., ** : p < 0.01). g Same experiment as in c, d, but testing the levels of nuclear SKP2 double mutant S72D/S75D instead of the tetramutant. h Quantification of n = 6 experiments as in g and represented as in f (T-test; SEM, *p < 0.05, ***p < 0.005)

false

• WB

CiteAb

Western blot - Anti-SIRT6 antibody (AB62739)

Western Blotting using Anti-SIRT6 antibody, ab62739. Publication image from Santos-Barriopedro, I. et al., 2018, Nat Commun, 29317652. Legend direct from paper.

SirT6 induces a modification in Suv39h1. a Western blotting of extracts from 293F cells transfected with Myc-Suv39h1 in the presence or absence of HA-tagged SirT1 or SirT6 (lanes 2 and 3, respectively). A SirT6-induced modification in Suv39h1 is indicated (red asterisk). b Endogenous Suv39h1 is also modified upon SirT6 upregulation. Suv39h1 Western-blot of extracts from 293 cells overexpressed or not with SIRT6-HA. c Western blotting of extracts from 293F cells expressing non-tagged Suv39h1 in the presence or absence of either Ubiquitin-HA15 and/or SirT6-HA. The effect of a titration of SirT6-HA (1, 3 and 6 µg transfected) on Suv39h1 (2 µg transfected) was tested (lanes 3–5). A lower exposition of the Suv39h1 main band is also shown. Red and blue asterisks indicate endogenous or HA-tagged ubiquitination in Suv39h1, respectively. d Quantification of the levels of modified Myc-Suv39h1 in the absence or presence SirT6-HA expression. Relative levels (%) of Suv39h1 modification compared to unmodified Suv39h1 are shown. The results were obtained from n = 3 replicas of experiment shown in lanes 1–2 of Fig. 4c. e Analysis, as in a, of Myc-Suv39h1 cotransfected with different HA-tagged SirT6 mutants. WT : SirT6 wild type; HY : H133Y; GA : G60A. f Schematics of the different Myc-tagged constructs of Suv39h1 used in g. g Western blotting with the indicated different Myc-Suv39h1 constructs − / + SirT6-HA. h Fractionation of 293F cells co-transfected with Myc-Suv39h1 and SirT6-HA. Nuclear extracts (NE) and nuclear insoluble pellet (NP) were generated using the Dignam method. NP was step-washed with increasing concentrations of NaCl (from 100–1000 mM). i Schematic summary of the experiment shown in j. j Fractionation of 293 cells transfected with Myc-Suv39h1 and − / + SirT1 or SirT6. Fractionation with the RIPA method generated a soluble fraction (RIPA, lanes 1–3) and a NP, which was further digested with Benzonase (lanes 4–6)

false

• WB

CiteAb

Western blot - Anti-SIRT6 antibody (AB62739)

Western Blotting using Anti-SIRT6 antibody, ab62739. Publication image from Santos-Barriopedro, I. et al., 2018, Nat Commun, 29317652. Legend direct from paper.

SirT6-induced monoubiquitination of Suv39h1 is induced by NF-κB pathway activation. a Suv39h1 levels from 293F cells transfected with Myc-Suv39h1 − / + SirT6-HA and analyzed at different degree of confluency. Cells were all plated at the same time and harvested at the indicated confluency (indicated in %). A quantification of n = 3 experiments is shown in Supplementary Figure 4a. b Western blotting of Myc-Suv39h1 − / + SirT6 expressed in 293F cells in the indicated conditions. C, control; DTB, double thymidine block; SS, serum starvation; NOC, nocodazole. A quantification of n = 3 experiments is shown in Supplementary Figure 4c. c Similar experiment as in b, with the indicated treatments. C, control; HU, hydroxyurea; CPT, camptothecin. FACS analysis of these treatments are included in Supplementary Figure 4b. A quantification of n = 3 experiments is shown in Supplementary Figure 5b. d SirT6 depletion by shRNA. Western blotting of endogenous SirT6 in 293F cells transfected with either scramble shRNA (Sc) or SIRT6 shRNA (Sh6). e Cells in d treated with the indicated conditions. A quantification of n = 3 experiments is shown in Supplementary Figure 5e. f Nuclear fractionation of Suv39h1 monoubiquitination induced by TNFα in 293F cells. Left panel, either total nuclear fraction or nuclear pellet digested with Benzonase (see online Methods) is shown. Right panel, quantification (n = 3) of the relative abundance of Suv39h1mUb vs total Suv39h1 in total nuclear fraction. (T-test; SEM, *p < 0.05, **p < 0.01). g Induction of Suv39h1mUb by TNFα in 293F cells expressing either scramble shRNA or shSIRT6. h Western blotting of extracts from 293F cells transfected with the indicated combinations of SirT6-HA, Myc-Suv39h1, and FLAG-RelA, and incubated in the presence or absence of TNFα before collecting the cells. i Co-immunoprecipitation experiments using anti-FLAG resin of extracts from 293F cells transfected with the indicated combinations of FLAG-RelA and Myc-Suv39h1 and in presence or absence of TNFα

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