FIG 1.

EB2 is an mRNP cap-associated protein. (A) Top, 100 μg of S7 nuclease-treated cytoplasmic HeLa cell extracts were incubated with an m⁷GTP cap affinity matrix. Cell extracts were incubated in the absence or presence of m⁷GpppG cap analog (lanes 3 to 6), and proteins retained on the column were analyzed by Western blotting. Input (lanes 1 and 2) corresponds to 50 μg of cytoplasmic cell extract. Bands shown in each panel correspond to the same Western blot but were cropped for simplicity. Bottom, EB2 protein purified from bacteria was incubated with an m⁷GTP cap affinity matrix, and proteins bound to the column (lane 4) or from the flowthrough (lane 6) were analyzed by Western blotting. (B and C) HeLa total cell extracts expressing Flag-tagged EB2 were subjected to immunoprecipitation (IP) using an anti-Flag M2 affinity gel in the absence (lanes 3 and 4) or presence (lanes 5 and 6) of RNase A, and associated proteins were analyzed by Western blotting. Input (lanes 1 and 2) corresponds to 1/10 of cell extracts.