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. Author manuscript; available in PMC: 2011 Jul 23.
Published in final edited form as: Clin Cancer Res. 2008 Dec 1;14(23):7614–7623. doi: 10.1158/1078-0432.CCR-08-1366

Fig. 2.

Fig. 2

Engineered overexpression of14-3-3σ. A, PANC-1cells were stably transfected with an empty vector (Sham) or with the full-length human 14-3-3σ cDNA that was tagged with HA, yielding clone1 (C1) and clone 7 (C7). Cell lysates (20 μg/lane) from sham and clones were probed with the anti-14-3-3σ antibody. B, comparison of14-3-3σ levels in PANC-1clones C1and C7 with the levels of 14-3-3σ expressed in parental BxPC3 and COLO-357 cells (20 μg/lane). A and B, HA-tagged 14-3-3σ protein is slightly shifted (top band) compared with endogenous14-3-3σ (bottom band). ERK-2 served as a loading control. C, levels of secreted 14-3-3σ in conditioned medium. Protein concentrates from conditioned medium (40 μg/lane) were subjected to immunoblotting with anti-HA and anti-14-3-3σ antibody. Whole-cell protein lysate from C1was used as a positive control for the detection of HA-tagged14-3-3σ protein in conditioned medium. The membrane was blotted for tubulin to ensure that cytoplasmic proteins were not being spuriously released in conditioned medium. The membrane was also stained with Ponceau red to verify equal loading of lanes. D, localization of14-3-3σ protein. Immunofluorescent staining of sham-transfected (Sham) and 14-3-3σ-transfected PANC-1cells (C7) was done with the anti-14-3-3σ antibody. Hoechst staining was used to visualize the nuclei. Representative of two independent experiments, with similar staining patterns observed in both experiments. Magnification, ×400.