Figure 2

The 69-kDa Ku86v in MM cell lines is present in the cytosolic, nuclear and membrane fractions and binds DNA. Having determined that MM cell lines constitutively express 69-kDa Ku86v, we next studied the subcellular location of Ku86v. Moreover, since CD40 triggering induces expression of 86-kDa Ku86 on the cell membrane of MM cells, we also investigated whether CD40 triggering affected the subcellular location Ku86v. Fresh whole cell (W), cytosolic (C), nuclear (N) and membrane (M) cell lysate fractions were obtained from sCD40L-triggered (5.0 μg/mL for 4 hrs) and resting RPMI 8226 MM cells, and subjected to normal SDS-PAGE (A). Full-length and variant forms of Ku86 were detected by western immunoblotting using S10B1 anti-Ku86 mAb. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Relative expression of 69-kDa Ku86v (normalized to weakest band) was determined using image densitometry and expressed in bar chart format. The presence of Ku86/Ku86v protein-DNA complexes were next detected using EMSA (B and C). Cytosolic (C), nuclear (N) or membrane (M) protein extracts (4.0 μg/sample) were first obtained from CESS (negative control), CD40-triggered (5.0 μg/mL sCD40L for 4 hrs) or non-CD40-triggered RPMI 8226 MM cell lines. Non-competitive binding of Ku86/Ku86v to DNA (B) was detected using a specific biotin end-labeled DNA probe (20.0 fmol/sample), native PAGE, and hrp-conjugated streptavidin chemiluminescence imaging. Full-length Ku86-DNA complexes are found at position I, whereas DNA complexes with the truncation variant of Ku86 are found at position II. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. To confirm the specificity of DNA binding, a competitive assay (C), in which a variable amount of cell lysate (up to 5.0 μg/sample) was mixed with a fixed amount (4.0 pmol/sample) of non-biotin-labeled DNA, was also performed in the same fashion. All experiments were repeated with the SGH-MM5 MM cell line (data not shown), showed similar results, and performed in triplicate.