Journal: Cell reports

Article Title: NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency

doi: 10.1016/j.celrep.2023.112372

Figure Lengend Snippet: (A–D) Immunoblotting analyses of acetylated lysine (AcK) and poly(ADP-ribose) (PAR) (A), immunofluorescence images of TUJ1 with AcK (B) or PAR (C), and enzymatic activity of SIRT1, SIRT2, PARP1 and PARP2 (D) in ATG5+/+ and ATG5−/− hESC-derived neurons (3 w).

Article Snippet: SIRT2 Activity Assay Kit , Abcam , Cat# ab156066.

Techniques: Western Blot, Immunofluorescence, Activity Assay, Derivative Assay

Journal: Cell reports

Article Title: NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency

doi: 10.1016/j.celrep.2023.112372

Figure Lengend Snippet: Key resources table

Article Snippet: SIRT2 Activity Assay Kit , Abcam , Cat# ab156066.

Techniques: Recombinant, Cell Culture, Knock-Out, Cytotoxicity Assay, CyQUANT Assay, Proliferation Assay, TUNEL Assay, Isolation, Quantitation Assay, Activity Assay, TALENs, Software

Journal: JCI Insight

Article Title: Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation

doi: 10.1172/jci.insight.166314

Figure Lengend Snippet: ( A ) GFP-K18-WT and GFP-K18-R90C lentivirus–transduced A549 cells were treated with DMSO or PN (5 μM, 48 hours). Acetylated proteins were immunoprecipitated using an anti-AcK antibody followed by blotting with an anti-K18 antibody. ( B ) GFP-K18-R90C lentivirus–transduced A549 cells were used for immunoprecipitation with an anti-GFP antibody then blotted with an anti-AcK antibody. An input blot is also shown. ( C ) Lysates from GFP-K18-WT/GFP-K18-R90C–transduced cells were blotted with antibodies against SIRT2 or acetylated α-tubulin (actin blot is included as loading control). The average relative intensity of the indicated bands from 3 separate experiments is included beneath each blot. * P < 0.05 using unpaired, 2-tailed Student’s t test (comparing R90C DMSO with PN). ( D ) The percentage activation of SIRT2 in the presence of PN, NMN (activator control), and NAM (inhibitor control). * P < 0.05 using the least significant difference test, compared with PN (= 0). ( E ) Immunofluorescent double staining of K18 and SIRT2 in A549 cells expressing K18-R90C treated with DMSO/PN. Scale bar: 50 μm. ( F ) Anti-SIRT2 (and control nonimmune IgG) immunoprecipitates were prepared from cells used in panel E , and then blotted with anti-SIRT2/anti-K18 antibodies. ( G ) Liver lysates from DMSO/PN-treated mice expressing K18-R90C were used for immunoprecipitation with an anti-AcK antibody (separate livers/lane) followed by immunoblotting with an anti-K18 antibody (input lysate was similarly blotted). n = 3 (DMSO group), n = 2 (PN group). ( H ) SIRT2 immunoprecipitates were prepared from liver extracts isolated from K18-R90C–expressing mice pretreated with DMSO/PN. K18 was analyzed by immunoblotting. Input lysates were also blotted with anti-SIRT2 (separate livers/lane are shown). n = 3 (DMSO group), n = 3 (PN). For panels G and H , the mean relative band intensity for each of the 2 groups is included beneath each blot. * P < 0.05, *** P < 0.001 using unpaired, 2-tailed Student’s t test (comparing DMSO with PN).

Article Snippet: The assay was performed according to the instructions of the human SIRT2 Direct Fluorescent Screening Assay Kit (Cayman Chemical).

Techniques: Immunoprecipitation, Activation Assay, Double Staining, Expressing, Western Blot, Isolation

Journal: JCI Insight

Article Title: Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation

doi: 10.1172/jci.insight.166314

Figure Lengend Snippet: ( A ) K18-R90C–transduced cells were treated with the SIRT2 inhibitor AGK2 (10 μM, ±PN, 48 hours). Images from the stained cells were quantified for the percentage cells with dots. P = 0.004, comparing DMSO vs. PN; P < 0.05, comparing PN vs. PN + AGK2. ( B ) Duplicate cells from panel A were then solubilized with Empigen and used for immunoprecipitation with an anti-AcK antibody followed by blotting of the precipitates/input lysate with anti-K18 antibody. Total lysates from duplicate cells were also blotted with the indicated antibodies. ( C ) K18-R90C–expressing cells were cultured with control or SIRT2 siRNA (24 hours) and then treated with DMSO/PN (48 hours). Images from stained cells were quantified for the percentage cells with dots. ( D ) Duplicate cells from panel C were used for immunoprecipitation and blotting as in panel B . Coomassie staining is included to show equal loading. Scale bars: 50 μm ( A and C ). All experiments were repeated at least 3 times with similar results. One-way ANOVA was used for comparison between treatment groups, and Tukey’s post hoc test was used for 2-group comparisons.

Article Snippet: The assay was performed according to the instructions of the human SIRT2 Direct Fluorescent Screening Assay Kit (Cayman Chemical).

Techniques: Staining, Immunoprecipitation, Expressing, Cell Culture