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. 2020 Dec 7;11:6249. doi: 10.1038/s41467-020-20069-4

Fig. 4. VDR interactants, intracellular localization, and transcriptional activities.

Fig. 4

a Volcano plot representing peptides identified by mass-spectrometry of proteins bound to cytosolic VDR in cells co-treated with 100 nM 1,25D3 and 1 µM ZK relative to vehicle-treated cells. n = 3 technical replicates/condition. The dashed line represents a p-value of 0.05. Source Data are provided as a Source Data file. b Abundance of WBP4 peptides determined by mass-spectrometry of VDR-immunoprecipitated cytosolic extracts of IEC-18 cells treated for 1.5 h with vehicle, 100 nM 1,25D3 or 100 nM 1,25D3 and 1 µM ZK. Cytosolic extracts from IEC-18 cells treated for 1.5 h with 100 nM 1,25D3 and 1 µM ZK immunoprecipitated with IgG were used as a negative control. n = 3 technical replicates/condition. c Representative immunoblot of VDR in WBP4-immunoprecipitated cytosolic extracts of IEC-18 cells treated for 1.5 h with vehicle, 100 nM 1,25D3 or 100 nM 1,25D3 and 1 µM ZK. Cytosolic extracts from IEC-18 cells treated for 1.5 h with 100 nM 1,25D3 and 1 µM ZK immunoprecipitated with Rabbit IgG were used as a negative control (left panel). *Indicates antibody light chains. Representative immunoblot of VDR, WBP4, and GAPDH (used as loading control) in cytosolic extracts of IEC-18 cells treated for 1.5 h with vehicle, 100 nM 1,25D3 or 100 nM 1,25D3 and 1 µM ZK (Input, right panel). Unprocessed blots in Source Data. d Representative immunostaining of WBP4 in IEC-18 cells treated for 1.5 h with vehicle, 100 nM 1,25D3, or with 100 nM 1,25D3 and 1 µM ZK (top panels). Superposition with DAPI-stained nuclei (bottom panels). Scale bar: 10 μm. n = 2 independent biological replicates/condition. e Representative VDR immunostaining in hVDR-transfected IEC-18 cells silenced (siWBP4) or not (siControl) for WBP4 (top panels). Superposition with DAPI-stained nuclei (bottom panels). Scale bar: 10 μm. n = 3 independent biological replicates/condition. f Transcript levels of Cyp24a1, S100g and Atp2b1 in IEC-18 cells silenced (siWBP4) or not (siControl) for WBP4. *p < 0.05 vs. siControl, Student’s t-test. n = 4 biological replicates/condition. g Transcript levels of Cyp24a1, S100g and Atp2b1 in IEC-18 cells transfected with WBP4 siRNA (siWBP4) and unrelated siRNA (siCtl) treated for 6 h with vehicle, 100 nM 1,25D3, or with 100 nM 1,25D3 and 1 µM ZK. n = 3 independent biological replicates/condition. *p < 0.05 vs. vehicle, one-way ANOVA with Tukey post-hoc test. Data in (b), (f) and (g) are represented as mean + s.e.m. The exact significant p-values are provided in Supplementary Table 4.