anti fas iii monoclonal  (Developmental Studies Hybridoma Bank)

Journal: Biochemistry and Biophysics Reports

Article Title: Mammalian fatty acid synthase and O -GlcNAc transferase preferentially interact via their respective N -terminal regions

doi: 10.1016/j.bbrep.2025.102427

Figure Lengend Snippet: FASN constructions used in this study. A. The delimitation of each domain is indicated by numbering the amino-acids. Each construction encodes a Flag-peptide located at the N-terminus. The domain boundaries shown are based on the UniProt primary sequence annotation (fatty acid synthase access number P49327 ). B. Domain boundaries shown are based on available experimental structures (PDB IDs: 3HHD , 8G7X , 4PIV , 4w82 and 7mhd ) ACP, Acyl Carrier Protein; DH, DeHydrogenase; ER, Enoyl-Reductase; KR, Keto-acyl Reductase; KS, Keto-acyl Synthase; MAT, Malonyl/Acetyl Transferase; TE, ThioEsterase.

Article Snippet: FASN gene was cloned into the pCMV10-3xFlag expression vector thanks to the Hin d III - FASN sense primer and FASN - Xba I antisense primer (Eurogentec) ( ).

Techniques: Sequencing

Journal: Biochemistry and Biophysics Reports

Article Title: Mammalian fatty acid synthase and O -GlcNAc transferase preferentially interact via their respective N -terminal regions

doi: 10.1016/j.bbrep.2025.102427

Figure Lengend Snippet: FASN and OGT interact via their respective N-terminal part. A and B. Hep3B cells were transfected with an empty vector (A) or with the different 3xFlag-FASN deletion mutants (B to G) in combination with OGT-HA overexpression. Protein samples were incubated with an anti-Flag antibody for co-immunoprecipitation with OGT (“IP” lanes for “Immuno-Precipitated”) ( panel A ) or with WGA-agarose beads (“P” lanes for “Purified”) ( panel B ). A. Co-immunoprecipitation of the deletion mutants with OGT analyzed by Western blotting. Optical densities of co-IP OGT were measured and normalized with the corresponding IP deletion mutants of FASN (red arrows; n = 3) (arbitrary units). B. O -GlcNAcylation levels of the deletion mutants analyzed by Western blotting. Specificity of WGA was checked by adding 0.5 M GlcNAc (“P + Gn” lane). Optical densities of the “purified” O -GlcNAcylated constructs were measured and normalized with inputs (red arrows; n = 3) (arbitrary units). ∗ p < 0.05; ∗∗ p < 0.01. C. Hep3B cells were transfected with 3xFlag-FASN. Protein samples were incubated with a GST-OGT construct (1–3) or GST only (4). After GST pull-down, interaction of the GST-OGT constructs (red arrows) with 3xFlag-FASN was analyzed by Western blotting. Optical densities of pull-down 3xFlag-FASN were measured and normalized with the corresponding construct. Interaction with GST only was subtracted from the other bands (n = 4). CD, Catalytic Domain; GST, Gluthatione-S Transferase; NLS, Nuclear Localization Signal; TPR, TetratricoPeptide Repeats. Dotted lines indicate where the blots were cut and joined during figure preparation. Molecular mass markers are indicated on the left (kDa).

Article Snippet: FASN gene was cloned into the pCMV10-3xFlag expression vector thanks to the Hin d III - FASN sense primer and FASN - Xba I antisense primer (Eurogentec) ( ).

Techniques: Transfection, Plasmid Preparation, Over Expression, Incubation, Immunoprecipitation, Purification, Western Blot, Co-Immunoprecipitation Assay, Construct

Journal: Biochemistry and Biophysics Reports

Article Title: Mammalian fatty acid synthase and O -GlcNAc transferase preferentially interact via their respective N -terminal regions

doi: 10.1016/j.bbrep.2025.102427

Figure Lengend Snippet: FASN and OGT interaction modeling. A. Modeling of FASN dimer and two OGT monomers; full sequences of the FASN dimer and of the OGT monomer were modeled independently with AlphaFold (see Materials and methods), assembled according to co-prediction performed in 3C and rendered in cartoon representation, FASN dimer in magenta and green (2511 residues each) and OGT monomers in cyan (1046 residues each). B. Same as A, but with FASN colored according to the domains depicted in and the four O -GlcNAcylated sites previously identified and reported, T315, S806, T980 and S1534, highlighted by red spheres . C. Model of FASN N -ter (bottom domain) and OGT N -ter (top domain, the TPR helices are clearly visible); the FASN sequence was trimmed to 1–969 and the OGT sequence to 1–286; the structure was predicted using AlphaFold3 and rendered in cartoon representation; residues are colored according to the pLDDT confidence score. D . Predicted Aligned Error matrix of the complex in C , ordered as OGT first and then FASN.

Article Snippet: FASN gene was cloned into the pCMV10-3xFlag expression vector thanks to the Hin d III - FASN sense primer and FASN - Xba I antisense primer (Eurogentec) ( ).

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: OGA inhibition positively impacts fatty acids synthesis . Cell lysates from HepG2 treated with the FASN inhibitor C75 or OGA inhibitor Thiamet-G (TG) were analyzed by Western blot (n = 3) according to their O -GlcNAc content ( A ). Optical densities were measured and normalized with GAPDH expression. Global ( B ) and individual ( C ) amounts of FAMES from HepG2-treated cells were measured by GC-FID (n = 6). Data are presented with means ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: To clone the FASN gene into the pCMV10-3xFlag expression vector, the Hind III - FASN sense (5' → 3') and FASN - Xba I antisense (3' → 5') primers (Eurogentec) ( ) were used to amplify the FASN sequence by PCR from the vCMVp-mCherry-FASN-V5Tag vector (eZyvec).

Techniques: Inhibition, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: Mutation of the T980 O -GlcNAcylation site reduces FASN expression, membrane association, homodimerization, and stability . Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors. Cell lysates were analyzed by Western blot (n = 8) by using an anti-Flag antibody ( A ). Molecular mass markers are indicated on the left (kDa). Optical densities were measured and normalized with β-actin expression. B , cytosolic and membrane-associated 3xFlag-FASN contents were prepared by cell fractionation and analyzed by Western blot (n = 3). Fractionation efficiency was assessed using anti-GAPDH and anti-E-Cadherin antibodies for cytosolic and membrane fractions, respectively. Optical densities were measured and normalized with these markers. C , cell lysates were analyzed by Native-PAGE (n = 3) according to their 3xFlag-FASN monomers (∼250 kDa) and dimers (∼500 kDa) contents. Optical densities were measured and normalized with total protein. D , CHX chase assay was performed by treating cells with 40 μg/ml CHX for the indicated time periods (0–24 h). Data are presented with means ± SD. ∗p < 0.05; ∗∗ p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: To clone the FASN gene into the pCMV10-3xFlag expression vector, the Hind III - FASN sense (5' → 3') and FASN - Xba I antisense (3' → 5') primers (Eurogentec) ( ) were used to amplify the FASN sequence by PCR from the vCMVp-mCherry-FASN-V5Tag vector (eZyvec).

Techniques: Mutagenesis, Expressing, Membrane, Transfection, Plasmid Preparation, Western Blot, Cell Fractionation, Fractionation, Clear Native PAGE

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: FASN T980 is crucial for FASN activity . Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors and incubated with the lipid droplet marker BODIPY 493/503 (n = 3) ( A ). Lipid droplets were counted using the ImageJ software ( B ). Confocal microscopy analysis indicates that FASN T980A mutation significantly impacts lipid droplets formation (bar size: 15 μm). Data are presented with means ± SD. ∗ p < 0.05.

Article Snippet: To clone the FASN gene into the pCMV10-3xFlag expression vector, the Hind III - FASN sense (5' → 3') and FASN - Xba I antisense (3' → 5') primers (Eurogentec) ( ) were used to amplify the FASN sequence by PCR from the vCMVp-mCherry-FASN-V5Tag vector (eZyvec).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Incubation, Marker, Software, Confocal Microscopy, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: FASN O -GlcNAcylation at T980 is pivotal for Hep3B cells survival, proliferation, and cell cycle progression. Low-density Hep3B cells were transfected with an empty vector or 3xFlag-FASN (wild type and mutants) expressing vectors. Six days later, colonies were fixed and stained with crystal violet (n = 3) ( A ). Using MTS assay ( B ), cell survival was evaluated according to densitometry measured at λ = 490 nm (n = 6). Cell proliferation was determined by cell counting (n = 4) ( C ). Cell cycle was analyzed by flow cytometry (n = 3) ( D ). Data are presented with means ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: To clone the FASN gene into the pCMV10-3xFlag expression vector, the Hind III - FASN sense (5' → 3') and FASN - Xba I antisense (3' → 5') primers (Eurogentec) ( ) were used to amplify the FASN sequence by PCR from the vCMVp-mCherry-FASN-V5Tag vector (eZyvec).

Techniques: Transfection, Plasmid Preparation, Expressing, Staining, MTS Assay, Cell Counting, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAcylation of fatty acid synthase is required for its proper subcellular localization, expression level, and activity

doi: 10.1016/j.jbc.2025.110497

Figure Lengend Snippet: FASN T980 is crucial for various properties of Hep3B cells. The O -GlcNAcylation at T980 is crucial for FASN expression, stability, membrane residence, homodimerization, and activity, promoting Hep3B cells survival, proliferation, and cell cycle progression. This highlights the O -GlcNAcylation of FASN at T980 as a potential key modification that supports hepatic carcinogenesis.

Article Snippet: To clone the FASN gene into the pCMV10-3xFlag expression vector, the Hind III - FASN sense (5' → 3') and FASN - Xba I antisense (3' → 5') primers (Eurogentec) ( ) were used to amplify the FASN sequence by PCR from the vCMVp-mCherry-FASN-V5Tag vector (eZyvec).

Techniques: Expressing, Membrane, Activity Assay, Modification