Figure 6: Analysis of intermediate filament proteins keratin 8/18.

(a) Analysis of intermediate filament proteins keratins 8 and 18 (a) Coomassie stained one-dimensional SDS–PAGE and western blot probed with fluorescent anti-keratin 8 and anti-keratin 18 antibodies. (b) Composite widefield fluorescence microscope images of HeLa (S) and HeLa/SN100 (R). Scale bars, 20 μm. (c) Western blot analysis of co-immunoprecipitation of the keratin 8/18 complex with and without crosslinking (XL) from drug-sensitive cells. Adding crosslinking to the IP stabilized the heterodimer and tetramer complexes. (d) Bar chart comparing relative quantitative values obtained from western blotting (n=6), fluorescence microscopy (n=4) and global SILAC (n=6) values for keratin 8 and keratin 18. Error bars represent 95% CIs for the mean values. (e) Crosslinked peptide network displaying the intra-crosslinked and inter-crosslinked peptides identified for keratin 8 and keratin 18. Nodes and edges are coloured the same as in Fig. 2, with red indicating increased levels in the chemoresistant cell line. Crosslinked lysine residue numbers are indicated for each node. Loop edges indicate unambiguous homodimeric crosslinked peptides. (f) Mapping of inter-protein crosslinked sites onto the domain structures for keratin 8 and 18. 7 observed crosslinked peptides are supportive of a parallel orientation between keratin 8 and keratin 18 while 8 crosslinked peptides are supportive of an antiparallel orientation. Full images of the blot and gel (a) are shown in Supplementary Fig. 10.