Anti flag antibody

After fixation by 4% formaldehyde for 15 min, cells were permeabilized with 0.1% saponin (Sigma, St. Louis, MO) for 15 min. Cells were washed three times in PBS and blocked in blocking buffer (1X PBS/5% normal serum/0.3% Triton™ X-100) for 60 min. Anti-FLAG antibody (1X PBS/1% BSA/0.3% Triton™ X-100) was applied at 1:500 (GenScript, Nanjing, China) and the incubation went overnight at 4 degree. After washing three times with PBS, the secondary PE-conjugated goat anti-mouse antibody (Abcam, Cambridge, UK, 1:500) was applied and incubated for 2 hours at room temperature. The cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO) and washed three times with PBS before they were sealed with nail polish and analyzed under a fluorescence confocal microscope (Nikon, Tokyo, Japan).

Clarified cell lysate was diluted in denaturing SDS gel loading buffer, and boiled for 15 min. After denaturing, the samples were loaded onto a 4 to 12% gel (GenScript, M41210C) for SDS-PAGE and separated by electrophoresis. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck-Millipore, ISEQ00010). The PVDF membrane was blocked with 5% skim milk in PBS containing 0.1% Tween 20, and then incubated with the following primary antibodies: anti-Flag antibody (GenScript, A00187), anti-Myc antibody (GenScript, A00172), anti-GST antibody (GenScript, A00097), or anti-His antibody (GenScript, A00174). The PVDF membrane was then washed three times with PBS and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (GenScript, A00160) and goat anti-rabbit antibody (GenScript, A00098). After three washes with PBST buffer, target protein bands were detected by using the enhanced chemiluminescence (ECL) reagent (Merck Millipore, WBLUR0500).

DNA corresponding to the sequences of the B. anthracisbrnQ1, brnQ2, brnQ3, brnQ4, brnQ5, and brnQ6 open reading frames was amplified from ANR-1 genomic DNA using PCR with the appropriate oligonucleotides shown in Table S1. To facilitate the detection of proteins encoded by these genes, amplification products carrying a sequence encoding a FLAG tag fused to the 3′ end of the gene were also generated. The purified PCR products were digested with the SalI and BamHI restriction enzymes and finally ligated into the shuttle vector pAW285 such that expression was dependent on the xylose-inducible promoter (70 (link)). As described above, recombinant plasmids were first generated in E. coli TG1 (Table 3) and subsequently transformed into an E. coli strain lacking dam and dcm to obtain unmethylated plasmid DNA for electroporation into B. anthracis. For the expression of the cloned genes, B. anthracis cultures were grown in CA-CO₃ to an OD₆₀₀ of ∼0.3 and induced with 1% xylose. After incubation for 3 h, cells were collected, and cell lysates were prepared as described previously (5 (link)). The production of FLAG-tagged transporter proteins was assessed by Western blotting using anti-FLAG antibody (GenScript, Piscataway, NJ, USA) as described previously (5 (link)).

The supernatant of cell lysates was diluted in denaturing buffer and boiled for 15 min. After denaturing, the samples were loaded onto a 4%–12% SDS-PAGE gel (Genscript) and separated by electrophoresis. Proteins were transferred to a PVDF membrane (Merck-Millipore, ISEQ00010). The PVDF membrane was blocked with 5% skim milk in PBS containing 0.1% Tween-20, and then incubated with the primary antibodies: anti-Flag antibody (1:1000, Genscript, A00187), anti-Myc antibody (1:1000, Genscript, A00172), or anti-GST antibody (1:1000, Genscript, A00097). Then, the membrane was washed three times with PBS and incubated with HRP-conjugated goat anti-mouse antibody (1:10,000, Genscript, A00160) and goat anti-rabbit antibody (1:10,000, Genscript, A00098). After three washes with PBST buffer, target protein bands were detected by using the enhanced chemiluminescence (ECL) reagent (Merck Millipore, WBLUR0500).