Isolation of native ATF5-containing protein complexes from rat C6 glioma cells
A) Verification of Flag-HA-ATF5 expression from the pCIN4-Flag-HA-ATF5 construct. In vitro translation (Promega) analysis of the pCIN4-Flag-HA-ATF5 showed that a 35 kd protein, consistent with the predicted size of double-tagged Flag-HA-ATF5, was produced as expected. pCIN4-Flag-HA-p53 (Positive Control) and pCIN4 (negative control) were used. Molecular markers (kd) are labeled on the right side. B) Selection of a C6 cell line that stably expresses Flag-HA-ATF5. pCIN4-Flag-HA-ATF5 or pCIN4 were transfected into C6 glioma cells and stable transfected cell lines were selected in growth medium containing G418 (0.8 mg/ml). Cell extracts were prepared from C6-pCIN4 or C6-pCIN4-Flag-HA-ATF5 cells and an equal amount (50 μg) of cell extracts from indicated cells was loaded for each lane and resolved by 10% SDS-PAGE. Immunoblotting (IB) analysis was carried out using indicated antibodies. Solid triangles indicate expression of Flag-HA-ATF5 (35 kd); open triangle indicates endogenously expressed ATF5 that is 22 kd (5). The difference in size (about 13 kd) between the ectopically expressed Flag-HA-ATF5 and the endogenous ATF5 is contributed by the Flag-HA-tag as well as by the apparent preference of the second potential in-frame Kozak start site that is used to produce the endogenous ATF5 in every type of cell tested so far (2, 5). The two translation start sites are separated by 100 amino acids, or about 11 kd. Molecular weights (kd) are labeled on the left side. C) Analysis of ATF5-containing complexes isolated by a dual-tag/double-immunoprecipitation (IP) strategy. Whole cell extracts from C6-pCIN4 and C6-pCIN4-Flag-HA-ATF5 cells were subjected to affinity chromatography on M2 (Flag antibody) agarose beads and an HA-affinity column. The bound proteins were eluted and examined for presence of Flag-HA-ATF5 bait, as described in Materials and Methods. A fraction of this eluate was re-IPed with anti-HA beads and subjected to 10% SDS-PAGE, followed with IB using an anti-Flag antibody. The Flag-HA-ATF5 in the elute gave a prominent band at 35 kd on the Western immunoblot.