Strain KK374/pKH441(hflD) was further transformed with the plasmids encoding the indicated model substrate. Cells were grown in the M9-based medium containing 1 mM IPTG and 1 mM cAMP at 30°C. An aliquot of the cells were sonically disrupted and fractionated into the soluble (S) and membrane (P) fractions (‘sonic’ samples). Another aliquot was treated with 0.1 N NaOH and fractionated into the supernatant (S) and pellet (P) fractions (‘NaOH’ samples). W indicates unfractionated whole cell samples. Proteins were analyzed by 12.5% SDS-PAGE and immunoblotting using anti-HA, anti-HflD, anti-FtsH, and anti-SecB antibodies. HflD, FtsH and SecB are peripheral membrane, integral membrane and cytosolic proteins, respectively, and served as controls for the fractionation. Because the chromosomally-encoded HflD is expressed at a very low level and hard to detect, we expressed HflD from a multicopy plasmid. The majority of the model substrates, HA-MBP-RseA(LY1)148, HA-MBP-YqfG and HA-MBP-YoaJ, was associated with the membrane both after sonical disruption and after alkali extraction, although higher proportions of HA-MBP-YoaJ was recovered in the soluble fractions as compared with the other two model substrates, indicating that most of the SMP-derived model proteins are integrally associated with the membrane.