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Fig. 1 | Journal of Biomedical Science

Fig. 1

From: Reciprocal deregulation of NKX3.1 and AURKA axis in castration-resistant prostate cancer and NEPC models

Fig. 1

AURKA associates with NKX3.1 and phosphorylates it. A AURKA phosphorylates NKX3.1 in vitro. Recombinant NKX3.1 was incubated with 6-His-AURKA-TPX2 complex for 30 min. The proteins were separated by SDS-PAGE and visualized by autoradiography. The top panel is the autoradiograph, while the lower panel is the corresponding Coomassie blue-stained gel. All assays were repeated at least three times. B NKX3.1 and AURKA bind each other in C4-2 cells. AURKA was immunoprecipitated and its association with NKX3.1 analyzed. IgG was used as the negative control, and NKX3.1 IP was used as a positive control. C NKX3.1 and AURKA bind each other in C4-2 cells. NKX3.1 was immunoprecipitated and its binding with AURKA was analyzed. IgG was used as the negative control, and AURKA IP was used as a positive control. D AURKA knockdown does not impact the subcellular localization of NKX3.1 in C4-2 cells. Immunofluorescence micrographs of C4-2 cells infected with either scrambled or AURKA shRNA followed by probing with NKX3.1 antibody (red). Nuclear counterstain is represented by DAPI (blue). (Scale bar = 20 μm). AURKA knockdown was confirmed using Western blot analysis. Images for control cells (having much lower NKX3.1 expression levels) were shown in enhanced gain to assist in visualization of the red signal. E AURKA inhibition does not alter the subcellular localization of NKX3.1 in C4-2 cells. Immunofluorescence images representing the subcellular distribution of NKX3.1 (red) in response to Alisertib in C4-2 cells. The blue channel represents DAPI for the nuclear counterstain. (Scale bar = 20 μm). Images for DMSO treated cells (having much lower relative NKX3.1 expression levels) were shown in enhanced gain to assist in visualization of the red signal. F AURKA depletion does not affect the subcellular localization of NKX3.1 in 22Rv1 cells. Immunofluorescence analysis of 22Rv1 cells with and without AURKA knockdown. Texas Red was used for probing NKX3.1 and DAPI (blue) is used for nuclear counterstain. (Scale bar = 20 μm). Western blot for confirmation of AURKA knockdown in 22Rv1 cells. G Inhibition of AURKA activity has no effect on the subcellular localization of NKX3.1 in 22Rv1 cells. Images obtained from immunofluorescence microscopy with red—NKX3.1, blue—DAPI. (Scale bar = 20 μm). The images for DMSO treated cells, that have much lower NKX3.1 expression levels than Alisertib treated cells, were shown in enhanced gain to assist in visualization of the red signal. H AURKA does not regulate NKX3.1 subcellular residence in C4-2 cells. Subcellular fractionation of NKX3.1 in C4-2 cells in response to knockdown of AURKA is in agreement with immunofluorescence analysis. I AURKA does not regulate NKX3.1 subcellular residence in 22Rv1 cells. J NKX3.1 does not regulate AURKA’s subcellular residence in C4-2 cells. Scale bar equals 20 µM. AURKA (green) and nucleus (blue). K NKX3.1 does not regulate AURKA’s subcellular residence in 22Rv1 cells. Subcellular localization of AURKA did not change when NKX3.1 was silenced. Scale bar equals 20 µM. L Subcellular fractionation of AURKA in C4-2 confirms immunofluorescence analysis. Actin and lamin A were used as controls for cytoplasmic and nuclear fractions, respectively. M Subcellular fractionation of AURKA in 22Rv1 cells in response to knockdown of NKX3.1 agrees with immunofluorescence analysis. All experiments were conducted at least three independent times

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