Figure 3 | Oncogene

Figure 3

From: Monomeric but not trimeric clathrin heavy chain regulates p53-mediated transcription

Figure 3

CHC833-1406 is a monomer. (a) CHC833-1406 lacking C-terminal trimerization domain does not interact with full-length CHC1-1675. H1299 cells were transfected with FLAG-tagged full-length CHC1-1675 and HA-tagged full-length CHC1-1675 or CHC833-1406. HA-tagged CHC proteins in cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with indicated antibodies. (b) CHC833-1406 does not form a homo-oligomer. FLAG-tagged CHC (CHC833-1675 or CHC833-1406) and HA-tagged CHC (CHC833-1675 or CHC833-1406) were ectopically expressed in H1299 cells, and each HA-tagged CHC protein in cell lysates was immunoprecipitated with anti-HA antibody, followed by immunoblotting with indicated antibodies. (c) CHC833-1406 is present as a monomer. Schematic representation of recombinant CHC protein used in crosslinking experiments (Top panel). Silver-staining pattern of crosslinked species from purified CHC proteins (CHC1073-1675, CHC1073-1406 and CHC833-1406) is shown in bottom panels. Recombinant CHC proteins used in this assay were expressed in E. coli and purified as described in Materials and methods. Purified CHC proteins were cross-linked with BS3 at a final concentration of 2 mM for 10 min at 30 °C, and the reaction was stopped by the addition of 1/5 volume of 1 M Tris–HCl (pH 6.8). Cross-linked species were resolved by 10% SDS–polyacrylamide gel electrophoresis and visualized by silver staining. Asterisk indicates a nonspecific band.

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