Anti flag m2 affinity gel slurry

U2OS cells were transduced with Prelamin A or EGFP (control) adenoviruses and whole cell lysates were obtained by sonicating cells in IP buffer for 10 s on ice and collecting supernatant after centrifugation at 1500×g for 5 min at 4 °C. ANTI-FLAG® M2 Affinity Gel slurry (Sigma) was added to 500 µg of lysates and IP buffer was added to a final volume of 1 ml. Samples were incubated at 4 °C, rotating for 2 h. Bead-protein complexes were washed 3x in IP buffer, and following the final wash, the pellet was resuspended in 0.01% bromophenol blue, 200 mM DTT, 4% SDS and 20% glycerol and heated at 100 °C for 10 min before centrifugation and western blot analysis of supernatant.

Cells were washed in ice-cold PBS and lysed in 400 μl of iced lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, mm EDTA, 1% Triton X-100, one protease inhibitor mixture tablet per 50 ml (cOmplete®, Roche Diagnostics)). The cells were scraped and collected into cooled microcentrifuge tubes. Cell lysates were centrifuged at 12,000 × g for 10 min at 4 °C. Supernatants were transferred into chilled tubes containing 40 μl of a 50% anti-FLAG M2 affinity gel slurry (Sigma), prepared as per the manufacturer's instructions. Following overnight incubation on a rotation wheel, at 4 °C, samples were centrifuged at 6,000 × g for 30 s to pellet the anti-FLAG M2 affinity gel-bound proteins, followed by three washes with 0.5 ml of lysis buffer. Samples were resuspended in 2× SDS sample buffer containing 100 μm maleimide.

U2OS cells were transduced with EGFP or UCLA as described above. Cell lysates were obtained by sonicating cells in IP buffer (10 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitors) and collecting supernatant after centrifugation. ANTI-Flag® M2 Affinity Gel slurry (Sigma) was added to 400 µg of protein and IP buffer was added to a final volume of 500 µl. Samples were incubated at 4°C, rotating for 2 h. Bead-protein complexes were washed 3x in IP buffer and finally the pellet was resuspended in 4x sample buffer, heated at 100°C for 10 min and analyzed by western blot.