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Fig. 2. Phosphorylation-induced disassembly of vimentin IFs in vitro and in vivo results in disassembled subunits with the same molecular mass categories. (A) Bacterially expressed human vimentin IF polymers were phosphorylated in vitro by PKA and the resultant disassembly of depolymerized subunits was assayed by glutaraldehyde crosslinking (GA; concentration range: 0-0.06%) of the vimentin supernatant fractions after 30 minutes centrifugation at 200,000 g. (B) The in vitro disassembly of vimentin was compared to that in vivo, by incubating BHK-21 cells in the presence of 50 nM cl-A for 30 minutes. The cells were treated with 1% Triton X-100 and the particulate material was pelleted by centrifugation at 200,000 g for 30 minutes. The supernatants were then subjected to glutaraldehyde crosslinking (same concentration range as above) followed by immunoblottting. The results indicate that the phosphorylation-mediated release of vimentin subunits also occurs as tetramers in vivo.
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