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. 2016 Oct 20;15(23):3251–3267. doi: 10.1080/15384101.2016.1242534

Figure 10.

Figure 10.

LDOC1 downregulates GNL3L-mediated NF-κB dependent transcriptional activity by modulating p65 expression. (A) HEK293T cells were co-transfected with Flag-GNL3L and increasing amounts of LDOC1-HA along with pGL4.32[luc2P/NF-κB-RE/Hygro] and pRL-TK-Renilla luciferase (internal control) plasmids. The NF-κB dependent transcriptional activity was analyzed by luciferase assay after 48 hrs of transfection. (B) The expression levels of Flag-GNL3L, LDOC1-HA and endogenous p65 were assessed by western blot analysis using specific antibodies. LDOC1 mediated inhibition of apoptosis is GNL3L-dependent. (C) HEK293T or AGS or MCF-7 or SiHa cells were co-transfected with Flag-GNL3L or LDOC1-HA in combination with cyclin D1-promoter luciferase and pRL-TK-Renilla luciferase plasmids. Luciferase assay was performed at the end of 48 hours of transfection to assess the cyclin D1 promoter dependent transcriptional activity. (D) Annexin-V binding assay was performed in HEK293T cells co-transfected with LDOC1-HA or vector along with GNL3L siRNA or control siRNA. (E) The expression of LDOC1-HA and the level of GNL3L knockdown was analyzed by western blot analysis using anti-HA and anti-GNL3L antibodies, respectively.