Figure 3

Actin bundling/cross-linking by actinin and parafibromin. (A) GST-parafibromin, and parafibromin separated from GST after thrombin cleavage was analyzed by SDS-PAGE and stained with Coomassie Blue. On the left are shown molecular weights of the protein standards in kilodaltons. Arrows show the bands corresponding to GST-parafibromin (upper arrow for the panel on the left) and parafibromin (lower arrow for the panel on the right). (B) G-actin was polymerized to F-actin. F-actin was incubated with rabbit skeletal muscle actinin, parafibromin (2 different preps) or GST (2 different preps) alone, or together as indicated, and centrifuged at 10,000 g for 15 minutes to sediment higher order structures of F-actin (bundles and/or cross-linked networks). The F-actin in the supernate (not bundled/cross-linked) and pellet (F-actin bundles/networks) fraction was analyzed by SDS-PAGE and stained with Coomassie Blue. Lane 10 (buffer) contains F-actin that was sedimented without any added GST, actinin or parafibromin, and therefore corresponds to the background level of F-actin bundles/networks in the pellet (similar background amount was also seen in the lane 6 and 7 marked GST). Increased actin, above background level, in the pellet fraction (lane 1, 2, 3, 4, 5, 8 and 9) indicates F-actin bundling and/or cross-linking.