human dlat recombinant protein  (Proteintech)

Journal: Nature Communications

Article Title: Mitochondrial KMT9 methylates DLAT to control pyruvate dehydrogenase activity and prostate cancer growth

doi: 10.1038/s41467-025-56492-8

Figure Lengend Snippet: a Subcellular fractionation analysis of PC-3M or DU145 cells with quantification of mitochondrial KMT9α percentage. b Immunofluorescence staining of PC-3M cells showing KMT9α, mitochondria (MitoTracker), and nuclei (DAPI). Scale bar: 5 μm. c Proteinase K protection assay for mitochondria isolated from PC-3M cells. TFAM (mitochondrial matrix, MM), CYCS (intermembrane space, IMS), and TOMM20 (outer mitochondrial membrane, OMM) served as controls for compartmental digestion. d Import of [ 35 S]-labeled KMT9α and KMT9β into mitochondria isolated from PC-3M cells. Δψ, membrane potential; PK, proteinase K. e Presence of KMT9α and KMT9β in mitochondrial extracts of diverse cancer and non-cancer cell lines was revealed by Western blot analysis using the indicated antibodies. f Schematic illustration of KMT9α interactome in PC-3M cells. g Co-immunoprecipitation using antibodies against either the N-terminus (N) or the C-terminus (C) of KMT9α showing the interaction between DLAT and KMT9α in mitochondrial lysates. (Input: 5% of total extract). h Schematic illustration of global metabolomics analyses in siCtrl or siKMT9α-treated PC-3M cells. i – l Relative abundance of pyruvate ( i ), NAD + ( j ), lactate ( k ), and acetyl-CoA ( l ) identified by global metabolomics analyses in siCtrl or siKMT9α-treated PC-3M cells. m Schematic showing pyruvate conversion to acetyl-CoA by PDC in mitochondria. n PDC activity in PC-3M cells expressing different KMT9α variants with or without endogenous KMT9α depletion. o Restoration of PDC activity in vitro. Extracts of PC-3M cells transfected with siCtrl or siKMT9α were supplemented with recombinant wildtype KMT9 heterodimer (rKMT9α/KMT9β) or catalytically inactive heterodimer (rKMT9α (N122A)/KMT9β). i – l , n , o Data are shown as box plots ( i – l n = 6 biological replicates; center line indicates median, box bounds show 25th and 75th percentiles, whiskers represent minimum and maximum values within 1.5 times the interquartile range) or mean + SD ( n , o n = 4 biological replicates). Statistical significance was determined by two-sided Welch’s two-sample t-test with false discovery rate (FDR) correction ( i – l ) or two-sided Student’s t-test ( n , o ). ( a – e , g , n , o ). All experiments were independently repeated at least three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Recombinant human DLAT protein was purchased from Sino Biological Inc.

Techniques: Fractionation, Immunofluorescence, Staining, Isolation, Membrane, Labeling, Western Blot, Immunoprecipitation, Activity Assay, Expressing, In Vitro, Transfection, Recombinant

Journal: Nature Communications

Article Title: Mitochondrial KMT9 methylates DLAT to control pyruvate dehydrogenase activity and prostate cancer growth

doi: 10.1038/s41467-025-56492-8

Figure Lengend Snippet: a Western blot analysis of PDC immunocaptured from mitochondrial fractions of siCtrl- or siKMT9α-treated PC-3M cells using anti-pan-methyllysine antibody. b Detection of DLAT K596me1 in mitochondrial fractions of DU145 cells treated with siCtrl or siKMT9α and transfected with expression plasmid for LacZ, KMT9α, or catalytically inactive KMT9α (N122A) by Western blot. Membranes were decorated with the indicated antibodies. TOMM20 served as loading control. c In vitro methylation of DLAT. PDC immunocaptured from DU145 cells was incubated with recombinant (r) KMT9α/β or rKMT9α (N122A)/KMT9β and S-adenosyl methionine (SAM). Methylation reactions were analyzed by Western blot using anti-DLAT K596me1 or anti-DLAT antibody. d Detection of DLAT K596me1, KMT9α, and KMT9β in mitochondrial extracts of indicated human specimens by Western blot. e Detection of DLAT K596me1 in nuclear, mitochondrial, or cytosolic fractions of DU145 cells transfected with siCtrl or siKMT9α and expression plasmid for LacZ, MTS-KMT9α, or NLS-KMT9α by Western blot. LMNA, TOMM20, and α-Tubulin served as controls for nuclear, mitochondrial, and cytosolic fractions, respectively. f , g Activity of PDC immunocaptured from extracts of PC-3M cells transfected with expression plasmid for LacZ, DLAT, DLAT (K596R), or DLAT (K547R) in combination with siCtrl or siDLAT. Knockdown efficiency and expression of exogenous DLAT proteins was verified by Western blot ( f ). Data represent mean + SD from 4 biological replicates. Statistical significance was determined by two-sided Student’s t -test. ( a – g ) All experiments were independently repeated at least three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Recombinant human DLAT protein was purchased from Sino Biological Inc.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Control, In Vitro, Methylation, Incubation, Recombinant, Activity Assay, Knockdown

Journal: Nature Communications

Article Title: Mitochondrial KMT9 methylates DLAT to control pyruvate dehydrogenase activity and prostate cancer growth

doi: 10.1038/s41467-025-56492-8

Figure Lengend Snippet: a – c Proliferation of DU145 cells transfected with siCtrl or siDLAT in combination with expression plasmid for LacZ ( a , b ), DLAT ( a ), or DLAT (K596R) ( b ) as indicated. Knockdown efficiency and expression of exogenous DLAT proteins was verified by Western blot ( c ). α-Tubulin served as loading control. d – i Proliferation of DU145 cells transfected with siCtrl or siKMT9α in combination with an expression plasmid for LacZ ( d – h ), KMT9α ( d ), MTS-KMT9α ( e , g ), NLS-KMT9α ( f , g ), or MTS-KMT9α (N122A), and NLS-KMT9α (N122A) ( h ). Knockdown efficiency and expression of exogenous KMT9α proteins was verified by Western blot ( i ). α-Tubulin served as loading control. j Proposed mechanism for KMT9-mediated control of mitochondrial functions in PCa cells. KMT9 monomethylates DLAT at K596, which is required for PDC activity and mitochondrial metabolism. KMT9 depletion abolishes DLAT K596 monomethylation, thereby impairing PDC activity and mitochondrial metabolism in PCa cells. k , l Free fatty acid (FFA) ( k ) and triglyceride ( l ) levels in DU145 cells transfected with siCtrl or siKMT9α in combination with expression plasmid for LacZ, NLS-KMT9α, MTS-KMT9α, or MTS-KMT9α (N122A). m , n Proliferation of DU145 ( m ) or HepG2 ( n ) cells transfected with sub-optimal concentrations of siCtrl or siKMT9α in the presence or absence of 100 µM SB204990 to detect potential effects of co-treatment. a , b , d – h , k – n , Data are presented as mea n ± SD ( a , b , d – h , m , n , n = 4 biological replicates) or mean + SD ( k , l , n = 3 biological replicates). Statistical significance was determined by a two-sided Student’s t-test ( a , b , d – h , k – n ). All experiments were independently repeated at least three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Recombinant human DLAT protein was purchased from Sino Biological Inc.

Techniques: Transfection, Expressing, Plasmid Preparation, Knockdown, Western Blot, Control, Activity Assay

Journal: Nature Communications

Article Title: Mitochondrial KMT9 methylates DLAT to control pyruvate dehydrogenase activity and prostate cancer growth

doi: 10.1038/s41467-025-56492-8

Figure Lengend Snippet: a , b Representative mouse prostates ( a ) and prostate/body weight ratio (mg/g) from Ctrl ( n = 6 mice), Pten / Smad4 KO ( n = 4 mice), and Pten / Smad4 / Kmt9α KO ( n = 5 mice) mice ( b ). c , d Multiplexed immunofluorescence showing MKI67 and PCNA ( c ) or DLAT, TOMM20, KMT9α and DLAT K596me1 ( d ) in prostate tissue from indicated genotypes. Scale bar: 50 μm. e , f Quantification of mitochondrial KMT9α/TOMM20 ( e ) and DLAT K596me1/DLAT ( f ) ratios in prostate sections (Ctrl n = 179 cells, Pten / Smad4 KO n = 404 cells, Pten / Smad4 / Kmt9α KO n = 182 cells). g , h Pearson correlation between DLAT K596me1/DLAT ratio and mitochondrial KMT9α/TOMM20 ratio ( g ) or nuclear KMT9α/DAPI ratio ( h ) in Pten / Smad4 KO prostate sections. R, Pearson correlation coefficient. i – k Analysis of PDC activity ( i ), free fatty acid levels ( j ), and triglyceride levels ( k ) in prostate tissue of Ctrl, Pten / Smad4 KO, and Pten / Smad4 / Kmt9α KO mice ( n = 6 mice per group). l , m H-score ratios of DLAT K596me1/DLAT ( l ) and mitochondrial KMT9α/TOMM20 ( m ) in normal ( n = 16 tissue microarray cores), normal adjacent tissue (NAT, n = 16 tissue microarray cores), and PCa tissues of Gleason grades 1-5 ( n = 18, 27, 10, 48, 52 tissue microarray cores respectively). n , o Pearson correlation between H-score ratios of DLAT K596me1/DLAT and mitochondrial KMT9α/TOMM20 ( n ) or nuclear KMT9α/DAPI ( o ) in prostate tissue sections of human patients. R, Pearson correlation coefficient. Original immunofluorescence images used for the quantification are shown in Supplementary Fig. . b , e , f , i – m , Data represent mea n ± SD in bar graphs ( b , i – k ) or box plots ( e , f , l , m ; center line: median; box bounds: 25th and 75th percentiles; whiskers: minimum and maximum values within 1.5 times the interquartile range). b , e – o Statistical significance was determined using two-sided Student’s t-test ( b , e , f , I , i–m ) or Pearson correlation analysis with two-sided p-values calculated based on the t-distribution with n-2 degrees of freedom ( g , h , n , o ). All experiments were independently repeated at least three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Recombinant human DLAT protein was purchased from Sino Biological Inc.

Techniques: Immunofluorescence, Activity Assay, Microarray

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Autoantibodies Against Dihydrolipoamide S-Acetyltransferase in Immune-Mediated Neuropathies

doi: 10.1212/NXI.0000000000200199

Figure Lengend Snippet: (A) Representative results of western blot (WB) showing the reaction of chronic inflammatory demyelinating polyneuropathy (CIDP) patients' sera to the extracted mouse brain proteins. (B) Protein samples from mouse brain tissues were untreated or treated with PNGaseF. Proteins were then immunoblotted against the representative human sera. IgG antibodies from anti-NF155 antibody–positive patients reacted only against N -glycosylated NF155. Contrarily, IgG antibodies from patients with CIDP with specific bands reacted with mouse brain homogenates regardless of N -glycosylation treatment. (C) The target antigens were immunoprecipitated from the extracted mouse brain proteins, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and silver stained. As controls, the IgG antibodies from patients with CIDP who do not harbor specific bands were used for immunoprecipitation. A protein band around 60–70 kDa (arrowhead) was specifically immunoprecipitated by the patients' IgG antibodies and identified as dihydrolipoamide S-acetyltransferase (DLAT) by mass spectrometry. Molecular weight markers are shown on the left in kilodalton. (D) The samples in subpanel (C) were analyzed by WB using anti-DLAT antibody.

Article Snippet: An enzyme-linked immunosorbent assay (ELISA) was performed to measure the anti-DLAT antibodies in the patient serum using recombinant human full-length DLAT protein (ProSpec, Rehovot, Israel) as the antigen, with modifications to the protocol detailed elsewhere.

Techniques: Western Blot, Glycoproteomics, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Staining, Mass Spectrometry, Molecular Weight

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Autoantibodies Against Dihydrolipoamide S-Acetyltransferase in Immune-Mediated Neuropathies

doi: 10.1212/NXI.0000000000200199

Figure Lengend Snippet: (A) Sera of patients with chronic inflammatory demyelinating polyneuropathy (CIDP) that showed reactivity to DLAT in ELISA were confirmed by western blotting with recombinant human full-length DLAT. An association was observed between ELISA and WB reactivity. (B) The reactivity of sera from patients with CIDP (n = 160) to DLAT was compared with that of sera from healthy controls (n = 26) and patients with immune-mediated sensory neuropathy (n = 58), MS (n = 47), Guillain-Barré syndrome (n = 27), and other neurologic diseases (n = 40) by ELISA. (C) Subclasses of IgG to DLAT were confirmed by ELISA, with IgG1 and IgG3 showing predominance. (D) Specificity was confirmed by quenching with patient serum using recombinant DLAT.

Article Snippet: An enzyme-linked immunosorbent assay (ELISA) was performed to measure the anti-DLAT antibodies in the patient serum using recombinant human full-length DLAT protein (ProSpec, Rehovot, Israel) as the antigen, with modifications to the protocol detailed elsewhere.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Autoantibodies Against Dihydrolipoamide S-Acetyltransferase in Immune-Mediated Neuropathies

doi: 10.1212/NXI.0000000000200199

Figure Lengend Snippet: Clinical Characteristics of Patients With CIDP With or Without Anti-DLAT Antibodies

Article Snippet: An enzyme-linked immunosorbent assay (ELISA) was performed to measure the anti-DLAT antibodies in the patient serum using recombinant human full-length DLAT protein (ProSpec, Rehovot, Israel) as the antigen, with modifications to the protocol detailed elsewhere.

Techniques:

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Autoantibodies Against Dihydrolipoamide S-Acetyltransferase in Immune-Mediated Neuropathies

doi: 10.1212/NXI.0000000000200199

Figure Lengend Snippet: (A) In human tissues, DLAT was widely expressed in large dorsal root ganglion (DRG) neurons and axons of the peripheral nerves and nerve roots (arrows), with little staining in the spinal cord anterior horn cells. Scale bar = 100 µm. (B) Strong staining was also observed in large DRG neurons of mouse DRG. Scale bar = 50 µm.

Article Snippet: An enzyme-linked immunosorbent assay (ELISA) was performed to measure the anti-DLAT antibodies in the patient serum using recombinant human full-length DLAT protein (ProSpec, Rehovot, Israel) as the antigen, with modifications to the protocol detailed elsewhere.

Techniques: Staining

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Autoantibodies Against Dihydrolipoamide S-Acetyltransferase in Immune-Mediated Neuropathies

doi: 10.1212/NXI.0000000000200199

Figure Lengend Snippet: (A) Immunohistochemical staining of MHC class 1 and DLAT in frozen muscle tissue from a patient with antimitochondrial antibody–positive myositis. Scale bar = 100 µm. (B) Immunofluorescence staining of human IgG and B7-1 in a sural nerve tissue from a patient with anti-DLAT antibody–positive CIDP and a disease control (Charcot-Marie-Tooth disease 1a). Scale bar = 50 µm. (C) Immunofluorescence staining of MHC class 1 and B7-1 in a sural nerve tissue from a patient with anti-DLAT antibody–positive CIDP and a disease control (Charcot-Marie-Tooth disease 1a). Scale bar = 50 µm. (D) Immunohistochemical staining of B7-1 and DLAT in a sural nerve tissue from a patient with anti-DLAT antibody–positive CIDP. Scale bar = 50 µm. CIDP = chronic inflammatory demyelinating polyneuropathy.

Article Snippet: An enzyme-linked immunosorbent assay (ELISA) was performed to measure the anti-DLAT antibodies in the patient serum using recombinant human full-length DLAT protein (ProSpec, Rehovot, Israel) as the antigen, with modifications to the protocol detailed elsewhere.

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Control

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Autoantibodies Against Dihydrolipoamide S-Acetyltransferase in Immune-Mediated Neuropathies

doi: 10.1212/NXI.0000000000200199

Figure Lengend Snippet: (A) The reactivity of autoantibodies against DLAT was confirmed using HEK 293 cells overexpressing the DLAT plasmid. The results showed that IgG from anti-DLAT antibody–positive patients bound to HEK 293 cells and colocalized with commercially available anti-DLAT antibodies, whereas IgG from the negative controls did not. Immunoabsorption with recombinant DLAT protein abolished reactivity, confirming the specificity of anti-DLAT antibodies in CIDP patients' sera. We performed this assay using sera from 3 patients with CIDP (patients 1, 2, and 4) and showed the representative result. Scale bar = 20 µm. (B) Serum from patients with CIDP with anti-DLAT antibodies mainly stained large DRG neurons in mouse DRG sections with being colocalized with DLAT immunostaining. Immunoabsorption with recombinant DLAT protein abolished reactivity, confirming the specificity of anti-DLAT antibodies in CIDP patients' sera. Scale bar = 50 µm. (C) Immunostaining of S100β immunostaining, a marker for large diameter neurons, and that of isolectin B4, a marker for small diameter neurons, in mouse DRG sections. Scale bar = 50 µm. The code of patients corresponds to eTable 3 ( links.lww.com/NXI/A974 ). CIDP = chronic inflammatory demyelinating polyneuropathy.

Article Snippet: An enzyme-linked immunosorbent assay (ELISA) was performed to measure the anti-DLAT antibodies in the patient serum using recombinant human full-length DLAT protein (ProSpec, Rehovot, Israel) as the antigen, with modifications to the protocol detailed elsewhere.

Techniques: Plasmid Preparation, Recombinant, Staining, Immunostaining, Marker

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Autoantibodies Against Dihydrolipoamide S-Acetyltransferase in Immune-Mediated Neuropathies

doi: 10.1212/NXI.0000000000200199

Figure Lengend Snippet: Sera from healthy controls (n = 4), patients with DLAT-negative CIDP (n = 4), and patients with DLAT-positive CIDP (n = 3) were added to cell culture medium. (A) LDH assay using sera from healthy controls, anti-DLAT antibody–negative and antibody–positive CIDPs. No significant differences were found between these groups. (B) WST-8 assay using sera from healthy controls, anti-DLAT antibody–negative and antibody–positive CIDPs. No significant differences were found between these groups. (C) Representative fluorescence micrograph of DRG neurons in culture; DRG neurons were immunostained with anti-Tuj1 antibody. Scale bar = 100 µm. (D) Quantitative analysis by ratio of mean neurite lengths. No significant differences were found between these groups. CIDP = chronic inflammatory demyelinating polyneuropathy.

Article Snippet: An enzyme-linked immunosorbent assay (ELISA) was performed to measure the anti-DLAT antibodies in the patient serum using recombinant human full-length DLAT protein (ProSpec, Rehovot, Israel) as the antigen, with modifications to the protocol detailed elsewhere.

Techniques: Cell Culture, Lactate Dehydrogenase Assay, Fluorescence