Vimentin Monoclonal Antibody (V9)

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	-Mouse (1:2000)

Protocol tips
-Mouse (1:2000)

Publication protocol

For protein extraction, 5 × 105 cells per well were plated onto six-well plates and transiently transfected with indicated expression plasmids. At 48 h after transfection, cells were incubated with or without the proteasome inhibitor MG132 (10 μM) for an additional 6 h before protein extraction and western blot analysis. Primary antibodies against Flag (M2, 1:1,000) and HA (3F10, 1:4,000) were used for protein detection. For IP, HEK293 cells transfected with the indicated expression plasmids were lysed in buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 5 μg ml−1 aprotinin, 1 μg ml−1 pepstatin, 1% Nonidet P-40, 1 mM EDTA and 0.25% deoxycholate). Total cell lysates (1,000 μl) were incubated overnight with 1 μg of anti-HA or anti-Flag antibody conjugated to agarose beads (Roche Molecular Biochemicals) at 4 °C. The beads were then washed with lysis buffer, and the immunoprecipitated protein complexes were resolved by 10% SDS–PAGE.

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