Figure S5.
Evolutionary conservation of Ak4 and effects of kinase-dead mutations in M0 macrophages. (A) Multiple sequence alignment of Ak4 orthologs from representative vertebrates is shown. The query Ak4 sequence was obtained via NCBI BLAST using house mouse (M, musculus) as the reference, with selected matches including human (H. sapiens, 90% sequence identity), striped hyena (H. hyaena, 90%), blue whale (B. musculus, 90%), domestic cattle (Bos taurus, 88%), Goode’s thornscrub tortoise (Gopherus evgoodei, 81%), American alligator (Alligator mississippiensis, 81%), red crowned crane (Grus japonensis, 82%), tiger rattlesnake (Crotalus tigris, 80%), and coelacanth (L. chalumnae, 74%). The secondary-structure annotation is displayed in the top row. Conserved regions within the NMP-binding domain are highlighted in green box, the LID domain in red box, and the P loop in yellow box. Mutated residues K18, G89, R122, N137, A166, and T199 are marked with triangles. (B) Mock, Ak4 WT, or kinase-dead Ak4 mutants were transduced into Ak4 KO BMDMs using lentiviral vectors. Mock-transduced WT BMDMs served as controls. Intracellular ATP levels were measured in lentiviral-transduced WT (mock) and Ak4 KO (mock, Ak4 WT, or kinase-dead mutant) M0 BMDMs (n = 3). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA. Data are representative of two independent experiments, and each point represents data from one mouse with two technical repeats. Panel A shows sequence alignment of A K 4 across multiple species, highlighting conserved regions, including the P-loop, N M P-binding domain, and L I D domain. Specific residues (e.g., K 18, R 122, N 137, A 166, T 199) are indicated as mutation sites analyzed in the study. Panel B shows a bar graph showing relative A T P levels in A K 4 knockout macrophages reconstituted with wild-type or mutant A K 4 constructs. Conditions include Control, Empty vector, W T, G 98 S, K 18 A, K 18 A slash R 122 A, R 122 A, N 137 A, T 199 A, K 18 A slash N 137 A slash T 199 A, R 122 A slash N 137 A, R 122 A sash T 199 A, and A 166 D. Bars show mean plus minus S D from two independent experiments, with each point representing one mouse (two technical replicates). A T P levels are normalized to the control.

Evolutionary conservation of Ak4 and effects of kinase-dead mutations in M0 macrophages. (A) Multiple sequence alignment of Ak4 orthologs from representative vertebrates is shown. The query Ak4 sequence was obtained via NCBI BLAST using house mouse (M, musculus) as the reference, with selected matches including human (H. sapiens, 90% sequence identity), striped hyena (H. hyaena, 90%), blue whale (B. musculus, 90%), domestic cattle (Bos taurus, 88%), Goode’s thornscrub tortoise (Gopherus evgoodei, 81%), American alligator (Alligator mississippiensis, 81%), red crowned crane (Grus japonensis, 82%), tiger rattlesnake (Crotalus tigris, 80%), and coelacanth (L. chalumnae, 74%). The secondary-structure annotation is displayed in the top row. Conserved regions within the NMP-binding domain are highlighted in green box, the LID domain in red box, and the P loop in yellow box. Mutated residues K18, G89, R122, N137, A166, and T199 are marked with triangles. (B) Mock, Ak4 WT, or kinase-dead Ak4 mutants were transduced into Ak4 KO BMDMs using lentiviral vectors. Mock-transduced WT BMDMs served as controls. Intracellular ATP levels were measured in lentiviral-transduced WT (mock) and Ak4 KO (mock, Ak4 WT, or kinase-dead mutant) M0 BMDMs (n = 3). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA. Data are representative of two independent experiments, and each point represents data from one mouse with two technical repeats.

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