Fig 9. IbpB modulates the association of IbpA with aggregates in vivo.

(A) Expression of IbpA and IbpB in constructed strains. E. coli MC4100 WT, ΔibpA, ΔibpB and ΔibpAB cultures grown in 30°C were subjected to heat shock (42°C, 10 min and 48°C, 5 min) and analysed with SDS-PAGE followed by western blot against IbpA and IbpB. (B) Localization of IbpA and IbpB in soluble and aggregated protein fractions. WT, ΔibpA and ΔibpB strains were heat-shocked as in A, followed by isolation of soluble and aggregated protein fractions as described in Methods. Obtained fractions were then analysed by SDS-PAGE and Western blot. (C) IbpB presence allows for IbpA removal from aggregates. Bacterial strains were heat-shocked as in A and allowed to recover at 30°C. Aggregated and soluble protein fractions were isolated from culture aliquots sampled at indicated time points and further analysed by SDS-PAGE Coomassie staining and Western blot using antibodies against IbpA or IbpB. Quantifications of total aggregated proteins (Aggr. prot.) and sHsps (both Western blot -Wb and Coomassie blue -Cb analysis) were plotted against recovery time.