Impact of HMGB1/TLR Ligand Complexes on HIV-1 Replication: Possible Role for Flagellin during HIV-1 Infection
Figure 4
HIV-1-infected patients exhibit elevated levels of flagellin-specific antibodies. (a) Approximately 1.8 μg of recombinant flagellin was twofold serially diluted (4 series) and resolved on 10–20% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and detected with immunoblot assay. Serum from HIV-1-infected or control subjects diluted 1 : 1000 was used as a primary antibody. Each panel is a separate immunoblot of the same recombinant flagellin detected with serum from HIV-1 patients (HIV 1, HIV 2, HIV 3, HIV 4) or control subjects (CS 1, CS 2). To confirm equal sample loading and protein transfer, immunoblotted membranes from HIV 1 and CS 2 were stripped off and reprobed with monoclonal antibody directed against flagellin (blots in the lower panel). The experiments were performed using sera from ten HIV-1-infected patients and four control subjects. These presented data are representative for all immunoblots. The position of recombinant flagellin protein is indicated with an arrow to the right. Numbers to the left depict positions of molecular mass markers (in kDa). (b) Recombinant flagellin (column 1) and lysates of flagellated or aflagellate E. coli (columns 3 and 4) were subjected to SDS-PAGE and immunoblotted using serum from HIV infected patient (column 1 and 3, diluted 1 : 1000) as primary antibody or polyclonal anti-flagellin antibody (columns 2 and 4, diluted 1 : 1000). The membrane in column 1 was stripped off and reprobed with polyclonal anti-flagellin antibody (column 2). The numbers above the figures indicate the amount of sample loaded in each well (in μL). MM, magic marker (Invitrogen); F+: flagellated; F−: aflagellate whole bacterial lysate; Pkl: polyclonal; Ab: antibody. Numbers to the left depict positions of molecular mass markers (in kDa).
