Figure 3

Western immunoblot to detect expression of surface protein under iron-limiting conditions. Bacteria were grown to stationary phase in RPMI. Cell wall associated proteins were solubilized with lysostaphin and separated on a 7.5% SDS-PAGE gel and detected with rabbit antibodies followed by HRP-conjugated protein A-peroxidase. (A). Newman wild-type, Newman clfA, Newman clfA clfB, Newman clfA isdA clfB, Newman clfA clfB sdrCDE, Newman clfA isdA clfB sdrCDE, Newman clfA isdA clfB sdrCDE (pCU1) and Newman clfA isdA clfB sdrCDE (pCU1clfB⁺). (B). Newman wild type, Newman clfA, Newman clfA isdA, Newman clfA isdA clfB, Newman clfA isdA sdrCDE, Newman clfA isdA clfB sdrCDE, Newman clfA isdA clfB sdrCDE (pCU1) and Newman clfA isdA clfB sdrCDE (pCU1isdAB⁺). (C). Newman clfA, Newman clfA sdrCDE, Newman clfA isdA sdrCDE, Newman clfA clfB sdrCDE, Newman clfA isdA clfB sdrCDE, Newman clfA isdA clfB sdrCDE (pCU1), Newman clfA isdA clfB sdrCDE (pCU1sdrC⁺), Newman clfA isdA clfB sdrCDE (pCU1sdrD⁺) and Newman clfA isdA clfB sdrCDE (pCU1sdrE⁺). The primary antibodies used were (A) rabbit anti-ClfB (B) rabbit anti-IsdA and (C) rabbit anti-SdrD B repeats.