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Fig. 5. Oxidative stress of nontransfected SW480 intestinal epithelial cells and SW480 cells stably expressing FLAG-tagged K8(G62C). Extracts were prepared from control cells incubated under normal growing conditions `C' or from cells subjected to oxidative stress `S' by treatment with 20 mM H₂O₂ for 1 hour. (A) Anti-FLAG immunoblot of proteins extracted from cells separated under nonreducing conditions. FLAG-tagged K8(G62C) was only detected in the transfected cultures and a putative K8 homodimer with an apparent molecular weight of just over 100 kDa was also detected in cells expressing K8(G62C) after oxidative stress. (B) Same samples as in (A) but run under reducing conditions: the high Mr band in K8(G62C) cells is no longer apparent as the disulphide bond in the homodimer has been reduced and all the K8 runs as monomers. (C) Immunoblotting extracts run under nonreducing conditions with monoclonal antibody M20 (detects K8 and K18) showed that endogenous K8 did not form putative homodimers in nontransfected cells. The monomeric FLAG-tagged K8(G62C) is detected by M20 as a weaker signal than endogenous K8 with an apparent molecular weight of 54 kDa in the unstressed transfected cells. The homodimeric FLAG-tagged K8(G62C) is also detected by M20 in the stressed cells.

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