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Journal: bioRxiv
Article Title: Cellular thermal shift assay of subcellular isolates for evaluating drug-membrane target interactions
doi: 10.64898/2026.02.03.703651
Figure Lengend Snippet: (A) Western blot image of MPC1 at 12 kD in the soluble fraction from 37 to 66°C of whole cells treated with vehicle control and UK5099. (B) The western blot was quantified using Image Lab.Relative intensities were used to quantify protein abundance normalized to 37°C. Curves fitted to the Boltzmann Sigmoid equation and demonstrate no statistically significant difference between the melting temperature of MPC1 in the control and UK5099-treated conditions (n = 1, N = 3, extra sum of squares F test, p = 0.91). (C) Western blot image of MPC at 12 kD in the soluble fraction of whole cell lysate treated with vehicle control and UK5099. (D) Quantified protein abundance in the soluble fraction fitted to the Boltzmann Sigmoid equation demonstrate no significant difference in the melting temperatures between control and UK5099-treated conditions (n = 1, N = 3, extra sum of squares F test, p = 0.82). Blot images are representative of means obtained from independent replicates.
Article Snippet: The membrane was blocked in 2% BSA for two days before being probed with 1:500
Techniques: Western Blot, Control, Quantitative Proteomics
Journal: bioRxiv
Article Title: Cellular thermal shift assay of subcellular isolates for evaluating drug-membrane target interactions
doi: 10.64898/2026.02.03.703651
Figure Lengend Snippet: (A) Western blot image of MPC1 in the soluble fraction between the temperatures of 37 and 66°C in DMSO and UK5099-treated conditions. (B) The western blot was quantified as described above. The DMSO samples were fitted to a Boltzmann Sigmoid curve and compared to the UK5099 samples (n = 1-2, N = 3, paired t-test assuming unequal variance, * = p < 0.05, ** = p < 0.01). (C) Western blot image of MPC2 in the soluble fraction between the temperatures of 37 and 66°C in DMSO and UK5099-treated conditions. (D) The western blots were quantified as above and the DMSO samples were fitted to a Boltzmann Sigmoid curve and compared to the UK5099 samples (n = 1-2, N = 3, extra sum of squares F-test, p < 0.05). Blot images are representative of means obtained from independent replicates.
Article Snippet: The membrane was blocked in 2% BSA for two days before being probed with 1:500
Techniques: Western Blot
Journal: bioRxiv
Article Title: Cellular thermal shift assay of subcellular isolates for evaluating drug-membrane target interactions
doi: 10.64898/2026.02.03.703651
Figure Lengend Snippet: (A) Schematic representing hypothesis that cellular lysis disrupts mitochondrial membrane integrity whereas with use of mitochondrial isolation it is preserved. (B) Western blot image of MPC1 in the soluble fraction between the temperatures 37 and 66°C in control versus UK5099 treated samples. (C) The samples were fitted to the Boltzmann Sigmoid curve, and no significant difference was observed in the MPC1 melting temperature between control and UK5099-treated conditions (n = 1-2, N = 3, extra sum of square F test, p = 0.7611). Blot images are representative of means obtained from independent replicates.
Article Snippet: The membrane was blocked in 2% BSA for two days before being probed with 1:500
Techniques: Lysis, Membrane, Isolation, Western Blot, Control
Journal: bioRxiv
Article Title: Cold-inducible GOT1 activates the malate-aspartate shuttle in brown adipose tissue to support fuel preference for fatty acids
doi: 10.1101/2024.11.18.623867
Figure Lengend Snippet: (A) Generation of BAT-specific Got1 knockout mice. Exon 2 of the mouse GOT1 gene was targeted for deletion by the LoxP/Cre system. (B) qPCR analysis for detection of Got1 transcripts in Got1 fl/fl and Got1 BATKO male mice exposed to 4°C for 7 days (n=6-7/group). (C) WB analysis for GOT1 in Got1 fl/fl and Got1 BATKO mice housed at 23°C or exposed to 4°C for 7 days. (D) Body temperature of Got1 fl/fl and Got1 BATKO female mice during cold exposure (n=7-8/group). Core body temperature was measured using a rectal thermometer at indicated times. (E) WB and qPCR of UCP1 in BAT from the same mice described in D. (F) Glucose tolerance test of cold exposed- Got1 fl/fl and Got1 BATKO female mice (n=7-10/group). (G) Measurement of [ 3 H]-2DG uptake by cold-activated BAT. (H) The NAD + /NADH ratio in BAT homogenates. (I-M) Measurement of body weight, energy expenditure, RER, food intake, and locomotor activity in Got1 fl/fl and Got1 BATKO male mice (n=6/group). Mice were placed in indirect calorimetric chambers and monitored during β-adrenergic stimulation of BAT with a β 3 AR agonist CL316243 (1mg/kg BW, daily). (N) Oxidation of 14 C-labeled palmitate or pyruvate in BAT homogenates. BAT was extracted from mice exposed to 4°C for 7 days. (O) qPCR analysis of genes involved in glucose uptake, glycolysis, and FA oxidation in cold-activated BAT (n=6/group). (P) Uptake of 14 C-labeled pyruvate by the mitochondria isolated from cold-activated BAT. (Q) Reduced phosphorylation of the PDH E1α subunit at Ser232 with a concurrent increase in PDH activity in Got1-deficient BAT. (R) Measurement of pyruvate-dependent mitochondrial respiration in BAT explants extracted from mice exposed to 4°C for 7 days. All data are presented as the Mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, # p <0.0001.
Article Snippet:
Techniques: Knock-Out, Activity Assay, Labeling, Isolation