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. 2023 Aug 11;30(10):2351–2363. doi: 10.1038/s41418-023-01205-1

Fig. 2. FBXL4 mediates ubiquitination and degradation of BNIP3 and BNIP3L.

Fig. 2

A 293 T cells were transfected with FLAG-BNIP3, FLAG-BNIP3L and increasing amount of Myc-FBXL4. The WCL were prepared for WB with the indicated antibodies. B WCL from HeLa cells transfected with FBXL4-specific siRNA or negative control (siNC) were prepared for WB with the indicated antibodies. FBXL4 KO cell lines were generated through LentiCRISPRv2 methods. The WCL from parental and FBXL4 KO HeLa cells were prepared for WB with the indicated antibodies. C RT-qPCR measurement of FBXL4, BNIP3 and BNIP3L mRNA expression in HeLa cells transfected with FBXL4-specific siRNA or siNC, Data are shown as means ± SD (n = 3). P values are calculated by the Two-way ANOVA test. *p < 0.05, **p < 0.01. D, E Representative IF images from parental and FBXL4 KO HeLa cells, stained with BNIP3 (or BNIP3L), HSP60 and DAPI. Scale bar, 10 μm. The relative intensity of HSP60, BNIP3 (or BNIP3L) and BNIP3L were quantified and shown in E. Data were shown as means ± SD (n = 50). P values are calculated by the Two-way ANOVA test. ****p < 0.0001. F–H WB analysis of the indicated proteins in the WCL of parental and FBXL4 KO HeLa cells pretreated with DMSO or MG132 (20 µM) for 5 h and then treated with cycloheximide (CHX, 50 μg/ml) and harvested at different time points. At each time point, the intensity of BNIP3 (G) and BNIP3L (H) was normalized to the intensity of Actin and then to the value at 0 h. P values are calculated by the Two-way ANOVA test. ****p < 0.0001. I, J WB analysis of the indicated proteins in vivo ubiquitination assays performed products and WCL from 293 T cells transfected with the indicated plasmids.