Precast sds page gel

Nuclear and cytoplasmic cell lysates were loaded onto precast SDS-PAGE gels (Invitrogen) and transferred onto PVDF membranes. Membranes were blocked with 5% skim milk and incubated overnight at 4°C with a polyclonal rabbit anti-Lmnb1 antibody (1:5,000; Abcam) or monoclonal mouse anti-Gapdh antibody (1:1,000; Abcam), followed by a HRP-conjugated secondary anti-rabbit or anti-mouse antibody (1:5,000; Chemicon). HRP-conjugated antibodies were detected using enhanced chemiluminescence reagents (Pierce) on a Kodak Imager (Kodak).

Whole cell lysate samples were prepared using Laemmli buffer, resolved in precast SDS-PAGE gels (Life Technologies), and transferred onto Immobilon-PSQ Membranes (Millipore). The membranes were incubated in buffers from the WesternBreeze Blocker/Diluent kit (Life Technologies) and probed with corresponding primary antibodies (S3 Table). Bound antibodies were detected by IRDye secondary antibodies using the Li-COR Odyssey Fc system (Li-COR).

The cell pellets and exosomes were lysed in RIPA lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton, 0.1% SDS, 5 mM EDTA) containing complete protease inhibitor cocktail, phospho-stop (Roche Diagnostics, Meylan, France), dithiothreitol (Sigma Aldrich, St. Louis, MO, USA) and vanadate (Life technologies, Carlsbad, CA, USA). Equal amounts of soluble protein (30 micrograms) were loaded in precast SDS-PAGE gels (Life technologies, Carlsbad, CA, USA) and transferred to PVDF membranes for probing with specific primary antibodies overnight at 4 °C and subsequently with horseradish peroxidase-conjugated secondary antibody. Detection of the protein bands was achieved using chemiluminescence (Life technologies, Carlsbad, CA, USA) and exposure on X-ray films (Kodak Rochester, NY, USA). The t-test was used for the comparisons of various measurements between control and experimental conditions as well as between densitometric data in Western blots. p value < 0.05 was considered statistically significant.