Figure 4: TXNIP molecules form interprotomer disulphide bonds between Cys63 and Cys247.
From: The structural basis for the negative regulation of thioredoxin by thioredoxin-interacting protein
(a,b) Co-immunoprecipitation assays were performed using lysates from 293 T cells transfected with combinations of FLAG-tagged (F), HA-tagged, and GST-tagged full-length TXNIP, T–TXNIP, or mutant TXNIP. Immobilized proteins on FLAG-agarose beads or glutathione beads were visualized by immunoblotting using anti-HA, anti-FLAG, or anti-GST antibodies. One percent of the WCL was used as the input. (c) Proteomic analysis of the interprotomer-interacting TXNIP molecules fractionated by SDS–PAGE under non-reducing conditions. The MS/MS spectrum shows the interprotomer disulphide bond between Cys63 and Cys247 identified as 54VLWMQGSQQCK64-240GNHISGTCASWR251. Doubly charged [M+2H]+ peptide ions at m/z 1353.64 were fragmented via higher-energy collisional dissociation. Matched peaks are shown in red. The ion types of matched peaks are written in red for b- and y-ions and blue for ions from C-S and S-S bond cleavages. Annotations used are: P, strand VLWMQGSQQCK; p, strand GNHISGTCASWR; B and Y, ions from P; b and y, ions from p; p+32, persulfide ion of p formed by C-S bond cleavage reactions. (d) The effect of TRX on the interaction between TXNIP molecules. Co-immunoprecipitation assays were performed using lysates from 293 T cells transfected with FLAG-tagged TXNIP, HA-tagged TXNIP and FLAG-tagged TRX. Immobilized proteins on HA-agarose beads were visualized by immunoblotting using anti-HA and anti-FLAG antibodies. (a,b,d) The results are representative of at least two independent experiments with similar results.
