FIG. 3.
Localization of T and RI chimeras. Subcellular fractions were prepared and analyzed by SDS-PAGE and Western blotting as described in Materials and Methods. (A) Cells carrying pZA-ssPhoAΦRICTDcmyc were grown in the absence (lanes 2 and 3) or the presence (lanes 4 and 5) of 1 mM azide for 10 min in advance of induction. Cells from these cultures were collected by TCA precipitation and centrifugation, resuspended in SDS-PAGE sample buffer, and subjected to SDS-PAGE and Western blotting using anti-RI antisera as the primary antibody. Lane 1, molecular mass standards; lanes 2 and 4, samples from uninduced cultures; lanes 3 and 5, samples from induced cultures. (B) Cells carrying the pZA-RINTDΦPhoA were induced, fractionated, and analyzed by SDS-PAGE and Western blotting using anti-PhoA as the primary antibody. Lane 1, molecular mass standards; lane 2, mature form of PhoA; lane 3, blank; lane 4, cells from an uninduced culture; lane 5, cells from an induced culture; lane 6, total cell lysate; lane 7, 1,000 × g pellet; lane 8, 1,000 × g supernatant; lane 9, 100,000 × g supernatant (soluble fraction); lane 10, 100,000 × g pellet (membrane fraction); lane 11, detergent-extractable (1% NP40) membrane fraction; lane 12, detergent-insoluble fraction. (C) Cells carrying pZA-ssPhoAΦTCTD were grown in the absence (lanes 2 to 6) or the presence (lanes 7 to 11) of 1 mM azide for 10 min in advance of induction, harvested, fractionated, and analyzed by SDS-PAGE and Western blotting using anti-T antisera as the primary antibody. Lane 1, molecular mass standards; lanes 2 and 7, uninduced cells; lanes 3 and 8, induced cells; lanes 4 and 9, cells after spheroplasting; lanes 5 and 10, spheroplasts; lanes 6 and 11, periplasm.