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. 2005 May 23;169(4):591–601. doi: 10.1083/jcb.200412139

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Copyright © 2005, The Rockefeller University Press

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Figure 2. — FIAT interacts with ATF4. (A and B) Yeast two-hybrid protein interaction assays. (A) Growth on two-minus selection media. (B) Growth on three-minus selection media. (A and B) position 1, FIAT “bait” + ATF4 “target;” position 2, negative control; position 3, positive control; position 4, FIAT “bait” + FIAT “target” without leucine zipper; position 5, FIAT “bait” + FIAT “target;” position 6, negative control. Note that on three-minus selection media only yeasts at position 1 (FIAT + ATF4) and position 3 (positive control) grow, demonstrating a positive interaction between FIAT and ATF4. The failure of yeasts to grow at positions 4 and 5 confirm that FIAT does not homodimerize. (C) Coimmunoprecipitation. Endogenous ATF4 was immunoprecipitated from ROS 17/2.8 osteoblastic cells using an anti-ATF4 antibody, and the immunoprecipitates were probed with the anti-FIAT antibody (left). Reciprocally, endogenous FIAT was immunoprecipitated from ROS 17/2.8 cells using the anti-FIAT antibody, and the immunoprecipitates were probed with an anti-ATF4 antibody (right). FIAT was coimmunoprecipitated with ATF4 (left, lane 3) and ATF4 was also coimmunoprecipitated with FIAT (right, lane 3). Protein A–Sepharose or unrelated antibodies failed to precipitate the target proteins (lanes 1–2).