anti got1  (Cell Signaling Technology Inc)

Journal: Journal of Virology

Article Title: African swine fever virus hijacks host pyrimidine metabolism to promote viral replication

doi: 10.1128/jvi.00985-25

Figure Lengend Snippet: Endogenous aspartate synthesis regulates ASFV replication. ( A ) Exogenous aspartate deficiency does not affect the replication of ASFV (normal culture medium containing 150 µM aspartate). ( B ) Schematic diagram of endogenous synthesis pathway of aspartate. ( C ) Detection of the effect of aminooxyacetic acid hemihydrochloride (AOA) on the expression of ASFV-P30 protein using Western blotting. ( D ) RT-qPCR analysis of ASFV-B646L mRNA expression in infected cells treated with GOT1 siRNA. ( E ) Western blot and virus titration assessing the effect of GOT1 siRNA on ASFV-P30 expression and infectious virus production. ( F ) Exogenous aspartate supplementation restores ASFV-P30 protein levels suppressed by GOT1 siRNA. ( G ) RT-qPCR analysis of ASFV-B646L mRNA expression in infected cells treated with GOT2 siRNA. ( H ) Detection of the effect of GOT2 siRNA on the expression of ASFV-P30 protein expression and virus titer. For all experiments, PAMs were infected with ASFV at MOI = 1, and cell lysates were collected at 24 hpi for Western blotting. Statistical significance is indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. = no significant difference ( P ≥ 0.05).

Article Snippet: The antibodies used in this study included ASFV-P30 mouse monoclonal antibody (produced by our laboratory), β-tubulin ( M20005 ; Abmart), GOT1 (14886-1-AP; Proteintech), and GOT2 (67738-1-Ig; Proteintech).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Infection, Virus, Titration

Journal: Journal of Virology

Article Title: African swine fever virus hijacks host pyrimidine metabolism to promote viral replication

doi: 10.1128/jvi.00985-25

Figure Lengend Snippet: Temporal GOT1–GOT2 reciprocity directs aspartate flux for ASFV replication. ( A, B ) Expression levels of GOT1 and GOT2 during ASFV infection at various time points. ( C, D ) RT-qPCR and Western blot analysis of changes in GOT1 expression after GOT2 knockdown by siRNA. ( E ) Exogenous nucleoside supplementation restores ASFV-P30 protein levels suppressed by GOT1 siRNA. Unless otherwise indicated, PAMs were infected with ASFV at MOI = 1, and samples were collected at 24 hpi for Western blotting, RT-qPCR, and viral titer analysis. Statistical significance is indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. = no significant difference ( P ≥ 0.05).

Article Snippet: The antibodies used in this study included ASFV-P30 mouse monoclonal antibody (produced by our laboratory), β-tubulin ( M20005 ; Abmart), GOT1 (14886-1-AP; Proteintech), and GOT2 (67738-1-Ig; Proteintech).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Knockdown

Journal: Journal of Virology

Article Title: African swine fever virus hijacks host pyrimidine metabolism to promote viral replication

doi: 10.1128/jvi.00985-25

Figure Lengend Snippet: Schematic diagram illustrating the mechanisms by which ASFV regulates nucleotide synthesis precursors through multiple pathways. ASFV reprograms host central carbon and nitrogen metabolism to fuel de novo pyrimidine nucleotide synthesis. First, ASFV activates the PPP, diverting glucose-derived flux to generate R5P, which is essential for nucleotide backbone synthesis. Second, ASFV upregulates the expression of the glutamine transporter SLC1A5, enhancing cellular glutamine uptake. Imported glutamine serves dual roles: (i) as a nitrogen donor for nucleotide biosynthesis, and (ii) as a carbon source, being converted to α-KG via glutaminolysis. α-KG enters the TCA cycle and is further converted by GOT1 to produce aspartate, which is required for pyrimidine ring formation. Together, these pathways ensure an adequate supply of nucleotide precursors to support ASFV DNA replication and gene expression. Viral hijacking of host metabolism thus represents a coordinated strategy to sustain robust viral replication.

Article Snippet: The antibodies used in this study included ASFV-P30 mouse monoclonal antibody (produced by our laboratory), β-tubulin ( M20005 ; Abmart), GOT1 (14886-1-AP; Proteintech), and GOT2 (67738-1-Ig; Proteintech).

Techniques: Derivative Assay, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Succinate Dehydrogenase loss causes cascading metabolic effects that impair pyrimidine biosynthesis

doi: 10.1101/2025.02.18.638948

Figure Lengend Snippet: Temporal measurements of aspartate levels and cell proliferation upon treatments that constrain aspartate acquisition reveals distinct patterns. a. Model depicting three methods of inducing aspartate limitation in cells. Complex I inhibition with rotenone blocks NAD+ regeneration thereby slowing TCA cycling; GOT1/2 double knockout (DKO) blocks transamination of oxaloacetate (OAA) to generate aspartate; and atpenin A5 (AA5) inhibits succinate dehydrogenase (SDH) activity, directly blocking the oxidative TCA cycle upstream of aspartate synthesis. b. GFP/RFP ratio of 143B jAspSnFR3/NucRFP cells treated with a rotenone titration, with one condition cultured with 1 mM pyruvate (+PYR), in DMEM without pyruvate (n=4). c. NucRFP counts per well of 143B jAspSnFR3/NucRFP cells treated with a rotenone titration, with one condition cultured with 1 mM pyruvate in DMEM without pyruvate (n=4). d. GFP/RFP ratio of 143B jAspSnFR3/NucRFP GOT1/2 double knockout (DKO) cells against a titration of environmental aspartate concentrations in DMEM without pyruvate (n=4). e. NucRFP counts of 143B jAspSnFR3/NucRFP GOT1/2 double knockout (DKO) cells against a titration of environmental aspartate concentrations in DMEM without pyruvate (n=4). f. Model showing that aspartate acquisition and consumption are basally matched at steady state until acquisition is inhibited by either disrupting synthesis through complex I impairment (Rotenone) in WT cells or by double knockout (DKO) of GOT1 and GOT2 DKO) and slowing uptake by environmental aspartate withdrawal (-ASP). Following aspartate acquisition impairments, aspartate levels decay while the rate of aspartate consumption for biosynthesis (measured as cell proliferation) is maintained. When the aspartate pool is depleted to the point that it slows aspartate consumption, cells enter a new steady state at a lower aspartate level, where acquisition and consumption are slowed, but once again matched. g. GFP/RFP ratio of 143B jAspSnFR3/NucRFP cells treated with an AA5 titration in DMEM (n=4). h. NucRFP counts of 143B jAspSnFR3/NucRFP cells treated with an AA5 titration in DMEM (n=4). i. NucRFP counts of 143B jAspSnFR3/NucRFP cells treated with either Veh or 5 µM AA5 with or without 20 mM aspartate in DMEM (n=3). Data are plotted as means ± standard deviation (SD) except g and h which are shown as means ± standard error of the mean (SEM).

Article Snippet: Membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween-20 (TBS-T) and incubated at 4°C overnight with the following antibodies: anti-GOT2 (Proteintech, 14800-1-AP, 1:1000), anti-GOT1 (Cell Signaling, 34423S, 1:1,000), anti-GFP (Sigma, 1:1000), anti-FH (Origene, TA500675S, 1:1000), anti-SDHB (Atlas, HPA002868, 1:1,000), anti-pChk1 (Cell Signaling, 2348S, 1:1000), anti-pChk2 (Cell Signaling, 2197S, 1:1000), anti-GAPDH (Cell Signaling, 5174S, 1:5000), and anti-Vinculin (Sigma, SAB4200729, 1:10,000).

Techniques: Inhibition, Double Knockout, Activity Assay, Blocking Assay, Titration, Cell Culture, Standard Deviation