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. 2023 Jan 17;133(2):e163498. doi: 10.1172/JCI163498

Figure 3. BMX directly binds 3βHSD1 and phosphorylates Y344.

Figure 3

(A) LNCaP cells overexpressing 3βHSD1 were treated with ibrutinib for 1.5 hours with or without DHEA for 0.5 hours. Pan-phospho-tyrosine (pTyr) was detected by immunoprecipitation and Western blot. (B and C) Cells overexpressing 3βHSD1 were treated with ibrutinib or zanubrutinib for 3 hours, and DHEA for 1 hour (B) or 2 hours (C). Phospho-3βHSD1-Y344 was detected by immunoprecipitation and Western blot. Blots run in parallel, contemporaneously, using identical samples are shown. (D) Cells with cooverexpression of GST-3βHSD1 and HA-BMX or vehicle were treated with DHEA for 1 hour. Phospho-3βHSD1-Y344 was detected by immunoprecipitation and Western blot. Actin blots, serving as loading controls, were run in parallel, contemporaneously using identical samples with other blots. (E) Cells overexpressing GST-3βHSD were transfected with siNT or 1 of 2 siRNA sequences against BMX; phospho-3βHSD1-Y344 was detected by GST pull-down and Western blot. 3βHSD1 blots, serving as loading controls, were run in parallel, contemporaneously using identical samples with other blots. (F) Cells with cooverexpression of GST-3βHSD and HA-BMX or HA-BMX-KD (kinase dead) were treated with DHEA for 1 hour. Phospho-3βHSD1-Y344 was detected by immunoprecipitation and Western blot. (G) GST-3βHSD or HA-BMX was purified from 293T cells; 3βHSD1-GST was dephosphorylated using phosphatase in vitro, followed by a kinase assay and Western blot. (H–J) 293T cells were transfected with 3βHSD1 or Y344F mutant with or without cooverexpressed HA-BMX. 3βHSD1 or 3βHSD1-Y344F mutant was immunopurified, and an NAD+ turnover assay was performed. Mean ± SEM represents combined data from 3 independent experiments. *P < 0.05, ***P < 0.001 (1-way ANOVA with Bonferroni’s multiple comparisons test). (K) GST-tagged and flag-tagged WT or Y344F-3βHSD1 were transfected into C4-2 cells, GST pull-down was performed, and flag-tagged 3βHSD1 was detected by Western blot. Cells were treated with DHEA for 2 hours. (L) GST-tagged and flag-tagged WT or Y344F-3βHSD1 were transfected into C4-2 cells, GST pull-down was performed, and flag-tagged 3βHSD1 was detected by Western blot. Cells were treated with DHEA for 2 hours; zanubrutinib (10 μM) treatment was 24 hours.