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. 2019 Oct 24;47(22):11623–11636. doi: 10.1093/nar/gkz1004

Figure 5.

Figure 5.

KDM4B binds SF3B3 in response to androgen-deprivation. (A) In situ PLAs were performed using antibodies against KDM4B and SF3B3 in VCaP cells cultured in FBS or CFBS plus enzalutamide (enz. 10 μM). PLA signals (red), representing interactions between KDM4B and SF3B3, were significantly upregulated in androgen-deprivation conditions. Nuclei were stained with Dapi. (B) Cell lysates from VCaP cells cultured under FBS, CFBS, CFBS + H89 were immunoprecipitated with anti-KDM4B antibody and western blotted with antibodies indicated. H89 abolished the interaction between KDM4B and SF3B3. (C) WB of indicated proteins in VCaP cells cultured under the conditions indicated. H89 downregulated AR-V7 expression that was upregulated by androgen-deprivation. (D) HEK293T cells were transfected without or with HA-PKA. Cell lysates were immunoprecipitated with anti-HA antibody and western blotted with anti-HA and anti-KDM4B antibodies. (E) Recombinant KDM4B protein was incubated without or with catalytically active PKA and analyzed by Phos-Tag gel followed by WB with antibody indicated. (F) AR-V7 minigene reporter assay with cell transfected with KDM4B, KDM4Bm, or KDM4B(S666A) (n = 4, mean ± SD), *P < 0.05. (G) Relative mRNA of AR-V7 and AR in VCaP cells transfected with KDM4B or KDM4B(S666A). (H-I) HEK293T cells were transfected with myc-SF3B3 (525–1077) together with HA-KDM4B (H) or HA-KDM4B WT or S666A (I), treated with or without PKA inhibitor H89 (H). Lysates were immunoprecipitated with anti-HA antibody and western blotted with indicated antibodies.