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. Author manuscript; available in PMC: 2023 Apr 19.
Published in final edited form as: Nature. 2022 Oct 19;610(7932):562–568. doi: 10.1038/s41586-022-05299-4

Fig. 3. Phosphorylated SMPD3 is more stable due to reduced ubiquitination-mediated degradation.

Fig. 3

a, Western blot analysis showing the stability of SMPD3 after the administration of cycloheximide (CHX, translation inhibitor) in SW480 cells transfected with WT or mutant SMPD3 (S209A, S209D). b, Mass spectrometry analysis of the ubiquitination intensity at K103 on SMPD3 with or without nicotine treatment. c, Total ubiquitination of SMPD3 in SW480 cells transfected with WT or K103R SMPD3-flag with or without nicotine treatment. d, K48 ubiquitination of SMPD3 in SW480 cells transfected with WT or K103R SMPD3-flag with or without nicotine treatment. e. K48 ubiquitination of SMPD3 in SW480 cells transfected with WT, 209A or 209A plus K103R SMPD3-flag without or with nicotine treatment. f, Western blot analysis showed phosphorylation levels, ubiquitination levels, and protein levels of SMPD3 in SW480 cells transfected with three SMPD3-flag plasmids (WT, S209A, S209A/K103R) and treated with or without nicotine. g, Diagram of the SMPD3 protein phosphorylation and ubiquitination process under normal and nicotine stimulated conditions. Created with BioRender. h, Representative ileal SMPD3 protein phosphorylation and ubiquitination levels in HFHCD-fed mice subjected to PBS, B. xylanisolvens colonization, nicotine water, nicotine water plus B. xylanisolvens colonization, and nicotine water plus nicX knock-out B. xylanisolvens colonization for 20 weeks. (n = 4 mice/group). i, Representative western blot of ileal SMPD3 phosphorylation, ubiquitination and protein levels in nonsmokers and smokers with low nicotine or high nicotine concentration in terminal ileum mucosa biopsies. Experiments were performed with 30 nonsmokers and 30 smokers (14 with low nicotine levels and 16 with high nicotine levels). For b-f, nicotine (1 μg/mL) treatment for 24 h. Experiments in a-f were performed three times independently.