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. 2010 Dec 2;5(12):e14198. doi: 10.1371/journal.pone.0014198

Figure 6. Radiolabeling with 3H-palmitic acid and subcellular localization of ElpS-3Flag.

Figure 6

(A) ElpS-3Flag radiolabelled with 3H-palmitic acid was detected by autoradiography and immunoblots with anti-Flag antibody in cellular fractions after immunoprecipitation with mouse anti-Flag antibodies (IP a-Flag) and in 100-fold concentrated supernatants from CB15N (WT), CB15N elpS::elpS-3flag (WT elpS::elpS-3flag), CB15N Δ(gspC-gspN) elpS::elpS-3flag (Δ(C-N) elpS::elpS-3flag) and CB15N ΔelpS (ΔelpS) grown in poor medium containing 50 mCi/ml 3H-palmitic acid. (B) Probable cleavage site in ElpS sequence at position 38 from NH2-terminus based on N-terminal sequencing of the extracellular isoform of ElpS-3Flag. (C) Detection of ElpS-3Flag in membrane fraction. Immunoblots with anti-Flag, anti-FliF (membrane marker) and anti-CcrM (cytoplasm marker) were done on total lysates (T), soluble fractions (cytoplasm, Cy) and insoluble fractions (membrane, M) from CB15N elpS::elpS-3flag (WT elpS::elpS-3flag), CB15N Δ(gspC-gspN) elpS::elpS-3flag (Δ(C-N) elpS::elpS-3flag) grown in poor medium. (D) Detection of ElpS-3Flag in outer membrane fraction. Immunoblots with anti-Flag were done on inner membrane (IM), outer membrane (OM) and soluble fractions (cytoplasm, Cy) from CB15N elpS::elpS-3flag (WT elpS::elpS-3flag), CB15N Δ(gspC-gspN) elpS::elpS-3flag (Δ(C-N) elpS::elpS-3flag) grown in poor medium.