Characterisation of a polycional antiserum againts ubiquitin and its use in the investigation of ubiquitin conjugate distribution in normal and stressed 3T3-LI cells

Antiserum against ubiquitin was used to identify ubiquitin conjugates in 3T3-LI cells, and to investigate changes in the amount and distribution of these conjugates in cells subjected to stress. Stress was induced by incubation with canavanine, an amino acid analogue, and by exposure of cells to temperatures of 43°C (heat shock). SDS-PAGE, followed by immunoblotting, produced no observable change in ubiquitin conjugation with the canavanine as used in this protocol.Heat shock, in a variety of different exposure protocols, appeared to pro-duce an increase in a wide spectrum of ubiquitin conjugates of molecular weight>70kd. The conjugates wereresent in the Triton-extracted cell residues, which are nuclear-cytoskeletal-enriched preparations and this fact is discussed in the light of evidence for a site for protein sequestration prior to degradation which localises to a nuclear-intermediate filament-enriched cell fraction. Indirect immunofluorescence was employed in an attempt to visualize changes in the distribution of ubiquitin in whole cells which had been exposed to stress. No changes were observed, and this is discussed in terms of the specificity of the antiserum for different forms of ubiquitin, and in the light of other work which was successfully used the anti-serum to visualize ubiquitin associated with intracellular inclusions. Experiments are proposed to investigate more fully the changes in ubiquitin conwugation and their likely meaning with respect to protein degradation. Modification of procedures for microscopy are proposed, in order to visualize changes in ubiquitin distribution in whole cells, and to assess whether any such changes have relevance to pathological processes involving ubiquitin.

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