Fig. S3.

SufB intein with native exteins undergoes N-terminal cleavage. (A) C-extein antibody blot. Full-length, His-tagged SufB was overexpressed and treated with H₂O₂ and DEA, similar to Fig. 3 C and D. The lysates were separated by 12% SDS/PAGE and transferred to Immuno-Blot PVDF (Bio-Rad). An anti–C-extein antibody was used to probe splicing products at 1:5,000 dilution. The blot shows N-terminal cleavage, which increases with 1 mM and 8 mM H₂O₂ and all concentrations of DEA treatment. Again, similar to Fig. 3 C and D, the highest concentrations of H₂O₂ and DEA cause precursor accumulation. The SufB lane is purified inteinless M. smegmatis SufB protein as size control for ligated exteins. Products detected are: precursor (P; 96 kDa), N-terminal cleavage product (NC, intein+C-extein; 66 kDa) and ligated exteins (LE; 56 kDa). (B) SufB (ligated extein) antibody blot. The same lysates from panel A were separated and probed using a SufB antibody at 1:5,000 dilution. N-terminal cleavage is seen at all concentrations of H₂O₂ and DEA, and as in A, precursor accumulation occurs at the highest concentrations for both treatments. The 66-kDa NC band was used for N-terminal cleavage quantitation. An additional N-terminal cleavage product, the N-extein (NC; 30 kDa), is seen with this antibody. Western blots were scanned on a Typhoon imager and quantified using ImageQuant. Polyclonal antibodies were produced in rabbits against purified SufB protein of M. smegmatis or a synthetic C-extein peptide CVEPIAKELPMEYALE by Covance. Because of differential reactivity with polyclonal antibodies, ratios of different species in the Western blots are arbitrary, unlike fluorescence quantitation.