Figure 3
From: BEX2 has a functional interplay with c-Jun/JNK and p65/RelA in breast cancer
Immunofluorescence to assess p65 nuclear localization, and validation of BEX2 overexpression and knock-down. (A) Western blot analysis and immunofluorescence (IF) to confirm BEX2 overexpression. MCF-7 cells were transfected with either a BEX2 expression construct or control vector. Forty-eight hours after transfections, BEX2 overexpression was assessed by western blot analysis using BEX2 rabbit polyclonal antibody at 1:200 dilution (top panel) and by IF using BEX2 antibody at 1:100 dilution (bottom panel). For IF staining Alexa-594 anti-rabbit secondary antibody was applied at 1:500 dilution. (B) The percentage of Nuclear-Only staining for p65 by IF in MCF-7 cells. Forty-eight hours after transfections cell treatments were carried out in the following groups: 1) control-vector, 2) control-vector + ceramide (C2) treatment at 10 μM overnight, 3) control-vector + BAY11-7082 (BAY11) at 5 μM overnight, 4) BEX2 overexpression (BEX2+), and 5) BEX2 overexpression + BAY11 treatment overnight. The following day, IF staining was carried out using anti-p65 primary and Alexa-594 anti-rabbit secondary antibodies at 1:200 and 1:500 dilutions, respectively. *, is for C2 or BEX2+ group vs control and **, is for BEX2+/BAY11 vs BEX2+. Error Bars: ± 2SEM. (C) Cellular localization of p65 by IF in the control, ceramide-treated, and BAY11-treated MCF-7 cells as explained in (B). (D) Cellular localization of p65 by IF following BEX2 overexpression (BEX2+) with and without BAY11 treatment as explained in (B). (E) BEX2 protein level by IF after BEX2 Knock-Down in MCF-7 cells. Anti-BEX2 rabbit primary and anti-rabbit Alexa-594 secondary antibodies were used at 1:100 and 1:500 dilutions, respectively. Left panel: control, right panel: BEX2 knock-down. (F) BEX2 knock-down (KD) efficiencies by RT-PCR for BEX2-siRNA duplexes in breast cancer cell lines MCF-7 (MCF-KD) and MDA-MB-231 (MDA-KD). BEX2 transcript level following knock-down was measured relative to the non-targeting siRNA control. The average fold changes for the two sets of siRNA duplexes are shown in each cell line. Error Bars: ± 2SEM.