anti rabbit ki 67  (Proteintech)

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

Journal: Gut Microbes

Article Title: Rotavirus rewires host cell metabolic pathways toward glutamine catabolism for effective virus infection

doi: 10.1080/19490976.2024.2428425

Figure Lengend Snippet: List of reagents and antibodies used for this study.

Article Snippet: 24. , GOT1 (E4A4O) Rabbit mAb , Cell Signaling , 34423.

Techniques: Protease Inhibitor

Journal: Gut Microbes

Article Title: Rotavirus rewires host cell metabolic pathways toward glutamine catabolism for effective virus infection

doi: 10.1080/19490976.2024.2428425

Figure Lengend Snippet: RV infection in HT-29 cells is associated with altered regulation of different enzymes of glutamine metabolism pathway; functional inhibition of which decreases rotavirus replication . (a) Schematic diagram of the glutamine metabolism pathway in host cells. (b) Protein level expression of enzymes involved in glutamine metabolism: glutaminase (GLS: 65 kda), glutamate dehydrogenase (GLUD: 54 kda)), glutamate oxaloacetate transaminase 1 & 2 (GOT1 and GOT2: 41 kda) and asparagine synthetase (ASNS: 64 kda) was quantified in whole cell lysates of HT-29 cells at 6, 12, 18 and 24 h.p.i. and compared to the un-infected controls. The protein expression levels were normalized with respect to the internal loading control, β-actin. RV-NSP5 expression was also assessed as a marker of virus infection. Compared to the mock-infected HT-29 cells, increased protein expression was observed for GLS, GOT2 and ASNS, at early time-points. (C-E) RV- SA11 infected cells were treated with the increasing concentrations of functional inhibitors of (c) GLS (CB-839 (10 - 20 μM) (D) GLUD ( R -162 (20,30 μM) and (E) AOAA (0,150 and 300 μM) 1 h post infection and maintained for 12 h in DMEM containing 5 mM glucose and 2 mM glutamine. Viral RNA transcripts (VP6) were quantified in infected and drug-treated cells after normalization with housekeeping gene, 18S. Each bar in the bar graph represents mean ± SD of a minimum of three replicates. Significant decrease in viral RNA was evident on treatment with inhibitors CB-839 ( p ≤ 0.05, F = 18.82, df = 2) and AOAA ( p ≤ 0.05, F = 45.16, df = 2). Whole cell lysates were also used for Western blotting to determine changes in viral proteins, VP6 and NSP5 normalized against loading control, β-actin. Reduced levels of RV-VP6 and NSP5 proteins were evident in cells treated with CB-839 and AOAA. For R162 treatment, the decline in RV-SA11 protein or transcripts were not as significant as observed on treatment with CB-839 or AOAA. (f) HT-29 cells were transfected with negative control siRNA (150 nM) or siASNS (75, 100 and 150 nM) and subsequently infected with RV-SA11 followed by incubation till 12 h.p.i. The relative transcript expression level of VP6 RNA was quantified after total cellular RNA isolation and qRT-PCR. Western blotting was used to visualize RV-SA11 VP6 and NSP5 proteins. siASNS transfection at 100 and 150 nM concentration significantly reduced RV-SA11 RNA ( p < 0.05, F = 18.78, df = 3) and protein synthesis. One way ANOVA followed by post-hoc Tukey’s test comparing all pair of columns was used to determine level of significance (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: 24. , GOT1 (E4A4O) Rabbit mAb , Cell Signaling , 34423.

Techniques: Infection, Functional Assay, Inhibition, Expressing, Control, Marker, Virus, Western Blot, Transfection, Negative Control, Incubation, Isolation, Quantitative RT-PCR, Concentration Assay

Journal: Gut Microbes

Article Title: Rotavirus rewires host cell metabolic pathways toward glutamine catabolism for effective virus infection

doi: 10.1080/19490976.2024.2428425

Figure Lengend Snippet: Aspartate and asparagine supplementation to glutamine depleted/inhibitor treated HT-29 cells with RV-SA11 infection substantially rescues virus replication . (a-e) HT-29 cells were infected with RV-SA11 (M.O.I. 3) and incubated for 12 h in media containing 5 mM glucose and 2 mM glutamine; 5 mM glucose with no glutamine and 5 mM glucose with 2 and 4 mM each of glutamate, alpha-ketoglutarate, aspartate, asparagine and oxaloacetate. Expression levels of RV-VP6 and NSP5 proteins were determined by immunoblotting with specific antibodies and the quantified relative fold change (with respect to the first lane) was calculated after normalization against the housekeeping protein, β-actin. The low level of RV proteins in glutamine depleted media was mostly restored on addition of 2 mM and 4 mM of aspartate or asparagine to the media (DMEM with 5 mM glucose). (f) Intermediates of the glutaminolysis pathway (4 mM) were added individually or in combination in RV infected cells maintained in glutamine depleted media and changes in expression of RV proteins was assessed by immunoblotting. Asparagine either solely or in combination with other intermediates like alpha-ketoglutarate, glutamate or oxaloacetate was able to maximally restore RV infection in glutamine depleted media. (G-I) HT-29 cells infected with RV-SA11 were treated with inhibitors/siRNA targeting GLS, GOT1/2 and ASNS. Rescue experiments were performed by addition of intermediates downstream to the step catalyzed by the enzymes targeted with their functional inhibitors and viral protein levels checked through immunoblotting RV-VP6 and NSP5 protein levels were found mostly restored on addition of aspartate or asparagine, either singly or in combination with other supplements.

Article Snippet: 24. , GOT1 (E4A4O) Rabbit mAb , Cell Signaling , 34423.

Techniques: Infection, Virus, Incubation, Expressing, Western Blot, Functional Assay

Journal: Cell Reports

Article Title: HIF1α-dependent uncoupling of glycolysis suppresses tumor cell proliferation

doi: 10.1016/j.celrep.2024.114103

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-GOT1 , Proteintech , Cat#14886-1-AP; RRID AB_2113630.

Techniques: Transduction, Recombinant, DC Protein Assay, Bicinchoninic Acid Protein Assay, Western Blot, Control, Modification, Transfection, Mass Spectrometry