Figure 6.

Newly synthesized ezrin transiently attaches to intermediate filaments. (A) Six-day CACO-2 monolayers were pulsed with [³⁵S]methionine-cysteine for 20 min and chased for various times (10–120 min). The Triton-insoluble cytoskeleton was separated by centrifugation and solubilized in 1% SDS. Then, the supernatant was diluted to 0.2% SDS, 1% TX-100 immunoprecipitated with anti-ezrin antibody, or a pool of aliquots (1/6 of each sample) of all the other samples was immunoprecipitated with nonimmune IgG (control), and analyzed by SDS-PAGE and PhosphorImager. (B) The Triton-insoluble cytoskeleton from cells pulsed and chased as described in A, homogenated by sonication, and the small multiprotein fragments were immunoprecipitated with anti-K8 (TROMA I) antibody first. This first immunoprecipitation was controlled with parallel nonlabeled cytoskeletal fragments assaying the eluates by immunoblot with an anti-K8 mouse mAb (left, K8 WB). Then, the material was eluted in 1% SDS, diluted to 0.2% SDS, 1% TX-100 and immunoprecipitated again with anti-ezrin antibody. Finally, the second eluate was separated by SDS-PAGE and analyzed by PhosphorImager (right-hand side panel). The arrows point at the 80-kDa band in a typical PhosphorImager image of four experiments. The apparent mass of the lower band is 64 kDa.