Figure 2.

Levels of total, phosphorylated NF-κB p65 (Ser311) and IκB-α in rat bone marrow (BM): 1—control; 2—prednisolone administration (5 mg/kg of b.w.); and 3—prednisolone and vitamin D₃ (1,000 IU/kg of b.w.) administration. Immunoblotting analysis of phosphorylated at Ser311, total NF-κB p65, and IκB-α in whole BM lysates: representative immunoblots are shown next to the bar charts (A) and quantified using β-actin as an internal control. The bar graphs of IκB-α (B), total NF-κB p65 (C), and phosphorylated at Ser311 (D) are presented as means ± SEM (n = 6/group). Immunoblotting analysis of NF-κB p65 subunit phosphorylated at Ser311 in cytoplasmic (E) and nuclear (F) lysates: representative immunoblots are shown above the bar charts and quantified using β-actin and lamin B1 as the loading controls for the cytoplasmic and nuclear fractions, respectively. The bar graphs of phosphoNF-κB p65 in cytoplasmic and nuclear lysates are presented as means ± SEM (n = 6/group),*p < 0.05 vs. control, ^#p < 0.05 vs. prednisolone administration. Immunocytochemical analysis of phosphoNF-κB p65-positive (green fluorescence) BM cells (G). Hoechst 33342 (blue fluorescence) was used for nuclear staining. Scale bars indicate 10 µm (magnification 100×). Red arrows show diffuse cytoplasmic distribution of phosphoNF-κB in the control and prednisolone + vitamin D₃ administration groups and the nuclear localization of phosphoNF-κB in the prednisolone group. Acquiring 3D model of phosphoNF-κB nuclear translocation in BM cells in prednisolone-administered rats: based on the series of pictures obtained by scanning (with a step of 0.32 µm) of single phosphoNF-κB-positive cells (at least 5, magnification 100×) the 3D model of phosphoNF-κB nuclear translocation (H) were build using Zeiss LSM Image Browser software.