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. 2022 Mar 21;298(5):101853. doi: 10.1016/j.jbc.2022.101853

Figure 6.

Figure 6

Metabolic differences between crPAICS::2×Strep-PAICS and crPAICS::PAICS-2×Strep. Metabolic profiling of crPAICS::2×Strep-PAICS and crPAICS::PAICS-2×Strep after trypsin treatment. A, crPAICS::PAICS-2×Strep shows reduced levels of all DNPB intermediates (FGAR, AIR, CAIR, SAICAR, and AICAR) and IMP when compared with crPAICS::2×Strep-PAICS. Ratio paired two-tailed t test was performed to compare the metabolite abundances; p-value: ‘∗’<0.05,‘∗∗’<0.01, ‘∗∗∗’<0.005, ‘∗∗∗∗’ <0.0005. B, Western blot analysis indicating crPAICS::2×Strep-PAICS and crPAICS::PAICS-2×Strep expressing similar levels of the enzymes that convert IMP to AMP (ADSS) or GMP (IMPDH, GMPS). C, crPAICS::PAICS-2×Strep (magenta squares) partitions IMP into GMP and AMP differently than crPAICS::2×Strep-PAICS (cyan circles). The difference between total isotope enrichment in GMP and AMP is significantly higher in crPAICS::PAICS-2×Strep than in crPAICS::2×Strep-PAICS, indicating that IMP is preferentially partitioned into guanine nucleotide synthesis in crPAICS::PAICS-2×Strep. The difference between the two PAICS rescue lines was consistent both upon 6 h as well as 8 h of isotope incorporation (values from three or four biological replicates are plotted, black line indicates median). Paired one-tailed t tests were performed on mean and values from three independent experiments shown above; p-value: ‘∗∗’ <0.01. AIR, 5-aminoimidazole ribotide; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; CAIR, 5-phosphoribosyl-4-carboxy-5-aminoimidazole; DNPB, de novo purine biosynthesis; FGAR, phosphoribosyl-N-formylglycineamide; IMP, inosine monophosphate; PAICS, phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase; SAICAR, phosphoribosylaminoimidazolesuccinocarboxamide.