Cellular Fractionation and Immunoblotting

All cells used in this study were obtained from the American Type Culture Collection (ATCC). HeLa (human cervical cancer, ATCC# CCL-13), HCT116 (human colorectal cancer, ATCC# CCL-247), HEK293 (adenovirus infected human embryonic kidney, ATCC# CRL-1573) and HS68 (normal HDF, ATCC# CRL-1635) cells grown as monolayers in 10 cm diameter dishes were washed in ice-cold phosphate buffer saline (PBS) pH 7.4, scraped from culture dishes on ice using a plastic cell scraper and collected in 1.5 ml micro-centrifuge tubes in 1 mL of ice-cold PBS. After centrifugation (a "pop-spin" for 10 sec in an Eppendorf table top microfuge), supernatants were removed from each sample and cell pellets were resuspended in 900 μL of ice-cold 0.1% NP40 (Calbiochem, CA, USA) in PBS and triturated 5 times using a p1000 micropipette (Gilson, WI, USA). 300 μL of the lysate was removed as "whole cell lysate" and 100 μL of 4 × Laemmli sample buffer was added to it, then kept on ice until the sonication step. The remaining (600 μL) material was centrifuged for 10 sec in 1.5 ml micro-centrifuge tubes and 300 μl of the supernatant was removed as the "cytosolic fraction". 100 μL of 4 × Laemmli sample buffer was added to this fraction and boiled for 1 min. After the remaining supernatant was removed, the pellet was resuspended in 1 ml of ice-cold 0.1% NP40 in PBS and centrifuged as above for 10 sec and the supernatant was discarded. The pellet (~20 μL) was resuspended with 180 μL of 1 × Laemmli sample buffer and designated as "nuclear fraction". Nuclear fractions and whole cell lysates that contained DNA were sonicated using microprobes (Misonix, NY, USA) at level 2, twice for 5 sec each, followed by boiling for 1 min. 10 μL, 10 μL and 5 μL of whole cell lysate, cytoplasmic and nuclear fractions, respectively, were loaded and electrophoresed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [12 (link)] and transferred to nitrocellulose membranes (Pall Life Sciences, FL, USA). Membranes were incubated with anti-pyruvate kinase (Santa Cruz, CA, USA) or anti-α-tubulin (Calbiochem, CA, USA) antibodies as cytoplasmic markers or anti-lamin A (Santa Cruz, CA, USA) or anti-nucleoporin (Santa Cruz, CA, USA) as nuclear markers after blocking with 3% bovine serum albumin in 0.1% tween 20-PBS (t-PBS). Membranes were washed with t-PBS followed by incubation with HRP-conjugated anti-rabbit or anti-mouse secondary antibody. After washing with t-PBS, target protein signals were detected by ECL (GE Healthcare, Buckinghamshire, UK) on Kodak X-ray film.