Journal: bioRxiv

Article Title: Validation of GCN5L1/BLOC1S1/BLOS1 Antibodies Using Knockout Cells and Tissue

doi: 10.1101/2023.07.21.550091

Figure Lengend Snippet: Quantitative PCR (qPCR) was used to confirm that GCN5L1 gene expression was absent in KO MEF cells. N = 3.

Article Snippet: The peptide immunogens used to generate each of the GCN5L1 antibodies (other than the Proteintech antibody, which was not reported by the manufacturer) are shown in .

Techniques: Real-time Polymerase Chain Reaction, Gene Expression

Journal: bioRxiv

Article Title: Validation of GCN5L1/BLOC1S1/BLOS1 Antibodies Using Knockout Cells and Tissue

doi: 10.1101/2023.07.21.550091

Figure Lengend Snippet: The epitopes of each of the antibodies used in this study mapped to human GCN5L1 (1-153 aa). The peptide immunogen sequence for the Proteintech antibody was not reported.

Article Snippet: The peptide immunogens used to generate each of the GCN5L1 antibodies (other than the Proteintech antibody, which was not reported by the manufacturer) are shown in .

Techniques: Sequencing

Journal: bioRxiv

Article Title: Validation of GCN5L1/BLOC1S1/BLOS1 Antibodies Using Knockout Cells and Tissue

doi: 10.1101/2023.07.21.550091

Figure Lengend Snippet: GCN5L1 protein was essentially undetectable in KO MEFs using the Covance and Sigma antibodies (A,C). In contrast, an immunoreactive band at the correct molecular weight for GCN5L1 was detected in validated KO MEFs using both the Proteintech and Santa Cruz antibodies (B,D). N = 3.

Article Snippet: The peptide immunogens used to generate each of the GCN5L1 antibodies (other than the Proteintech antibody, which was not reported by the manufacturer) are shown in .

Techniques: Molecular Weight

Journal: bioRxiv

Article Title: Validation of GCN5L1/BLOC1S1/BLOS1 Antibodies Using Knockout Cells and Tissue

doi: 10.1101/2023.07.21.550091

Figure Lengend Snippet: Quantitative PCR (qPCR) was used to confirm that GCN5L1 gene expression was absent in KO liver tissue. N = 3

Article Snippet: The peptide immunogens used to generate each of the GCN5L1 antibodies (other than the Proteintech antibody, which was not reported by the manufacturer) are shown in .

Techniques: Real-time Polymerase Chain Reaction, Gene Expression

Journal: bioRxiv

Article Title: Validation of GCN5L1/BLOC1S1/BLOS1 Antibodies Using Knockout Cells and Tissue

doi: 10.1101/2023.07.21.550091

Figure Lengend Snippet: GCN5L1 protein was essentially undetectable in KO livers using the Covance and Sigma antibodies (A,C). In contrast, an immunoreactive band at the correct molecular weight for GCN5L1 was detected in validated KO livers using the Proteintech antibody (B). N = 3.

Article Snippet: The peptide immunogens used to generate each of the GCN5L1 antibodies (other than the Proteintech antibody, which was not reported by the manufacturer) are shown in .

Techniques: Molecular Weight

Journal: bioRxiv

Article Title: Validation of GCN5L1/BLOC1S1/BLOS1 Antibodies Using Knockout Cells and Tissue

doi: 10.1101/2023.07.21.550091

Figure Lengend Snippet: GCN5L1 protein was not detectable in either wildtype (WT) or knockout (KO) liver tissue using the Santa Cruz antibody. N = 3.

Article Snippet: The peptide immunogens used to generate each of the GCN5L1 antibodies (other than the Proteintech antibody, which was not reported by the manufacturer) are shown in .

Techniques: Knock-Out

Journal: Nature Communications

Article Title: Acetylation of C/EBPα inhibits its granulopoietic function

doi: 10.1038/ncomms10968

Figure Lengend Snippet: ( a ) Western blot analysis with anti-pan-acetyl-lysine antibody following immunoprecipitation (IP) of C/EBPα with rabbit anti-C/EBPα in HL-60 and Molm-14 human myeloid cell lysates. Rabbit anti-GFP antibody was used as IP control. ( b , c ) GCN5 decreases the ability of C/EBPα to transactivate a minimal p(CEBP) 4 TK promoter in a dose-dependent manner and is dependent on the GCN5 histone acetyltransferase (HAT) domain. 293T cells were transiently transfected with p(CEBP) 4 TK, pRL-null, and pcDNA6 expression plasmids for C/EBPα and with GCN5 or GCN5 (-HAT), respectively. Protein expression of corresponding constructs were shown in . Luciferase activity was measured in duplicate for each experiment and data are shown as mean±s.d. ( N =3). ( d ) Knockdown of endogenous GCN5 results in an increase in C/EBPα transactivation potential. GCN5 knockdown in 293T cells enhanced C/EBPα transactivation capacity on a minimal p(CEBP) 4 TK promoter with pRL-null and pcDNA6 C/EBPα plasmids. Firefly luciferase readings were normalized against internal control renilla luciferase. Luciferase activity was measured in duplicate for each experiment and data are shown as mean±s.d. ( N =3). ( e ) Western blot demonstrating knockdown efficiencies of endogenous GCN5 by 3 different shRNAs in 293T cells used in d . ( f ) GCN5 acetylates C/EBPα at K298, K302 and K326. C/EBPα peptides were incubated with the GST-HAT domain of GCN5 in the presence of 3 H-labeled acetyl-CoA. Tritium incorporation by C/EBPα peptides was measured by scintillation counting. Error bars represent mean±s.e.m. Experiments were performed twice in duplicate. ( g ) Structure of the C/EBPα basic region-leucine zipper domain bound to DNA. Arrows indicate the location of the acetylated lysine residues. ( h ) Endogenous C/EBPα is acetylated in the basic leucine zipper region in HL-60 and Molm-14 cells. C/EBPα protein was immunoprecipitated using a rabbit antibody recognizing total C/EBPα protein, followed by immunoblotting against acetylated C/EBPα antibodies (K298, K302, and K326). Rabbit anti-GFP antibody was used as a control for IP. ** P <0.01 and *** P <0.001; Student's unpaired t -test ( b , d and f ).

Article Snippet: GST-tagged recombinant GCN5 (Catalogue #K311-381G) was also purchased from SignalChem.

Techniques: Western Blot, Immunoprecipitation, Transfection, Expressing, Construct, Luciferase, Activity Assay, Incubation, Labeling

Journal: Current Research in Physiology

Article Title: Increased fatty acid oxidation enzyme activity in the hearts of mice fed a high fat diet does not correlate with improved cardiac contractile function

doi: 10.1016/j.crphys.2020.11.001

Figure Lengend Snippet: Physical characteristics of WT and cardiac-specific GCN5L1 KO mice. A 24 week HFD led to significant increases in ( A ) body weight and ( B ) heart weight in both WT and GCN5L1 KO mice. There was no change between genotypes in heart weight relative to body weight ( C ), suggesting that cardiac size changes are independent of GCN5l1 expression levels. N ​= ​5–10, ∗ ​= ​ P < 0.05 vs. WT LFD.

Article Snippet: GCN5L1 antibody was made by Covance and previously validated ( ; ).

Techniques: Expressing

Journal: Current Research in Physiology

Article Title: Increased fatty acid oxidation enzyme activity in the hearts of mice fed a high fat diet does not correlate with improved cardiac contractile function

doi: 10.1016/j.crphys.2020.11.001

Figure Lengend Snippet: Loss of GCN5L1 expression prevents HFD-induced fatty acid oxidation enzyme hyperacetylation. The acetylation status of ( A, B ) LCAD and ( C, D ) SCAD was significantly increased in the hearts of WT mice, but not GCN5L1 KO mice, after a 24 week HFD. N ​= ​4–5, ∗ ​= ​ P < 0.05 vs. WT LFD.

Article Snippet: GCN5L1 antibody was made by Covance and previously validated ( ; ).

Techniques: Expressing

Journal: Current Research in Physiology

Article Title: Increased fatty acid oxidation enzyme activity in the hearts of mice fed a high fat diet does not correlate with improved cardiac contractile function

doi: 10.1016/j.crphys.2020.11.001

Figure Lengend Snippet: Fatty acid oxidation activity is increased in isolated cardiac lysates from HFD-fed WT mice. The enzymatic activity of ( A ) LCAD and ( B ) SCAD was significantly increased in cardiac lysates from WT mice, but not GCN5L1 KO mice, after a 24 week HFD. N ​= ​5–10, ∗ ​= ​ P < 0.05 vs. WT LFD.

Article Snippet: GCN5L1 antibody was made by Covance and previously validated ( ; ).

Techniques: Activity Assay, Isolation

Journal: Current Research in Physiology

Article Title: Increased fatty acid oxidation enzyme activity in the hearts of mice fed a high fat diet does not correlate with improved cardiac contractile function

doi: 10.1016/j.crphys.2020.11.001

Figure Lengend Snippet: Increased fatty acid oxidation activity in HFD-fed WT mice does not result in improved cardiac function ex vivo . WT mice fed a 24 week HFD did not demonstrate improved cardiac ( A ) contractility or ( B ) relaxation relative to GCN5L1 KO mice under the same conditions. N = 4–8.

Article Snippet: GCN5L1 antibody was made by Covance and previously validated ( ; ).

Techniques: Activity Assay, Ex Vivo