Figure 4.

The interaction between CsdA and Hfq is RNA mediated. (A) Far western-blotting does not reveal an interaction between CsdA and Hfq. 25 pmol of CsdA-His₆ (lane 1) or 25 pmol of CsdA-His₆ (lanes 2) treated with micrococcal nuclease (MN) and 25 pmol of Hfq (lane 3, control) were separated by SDS-polyacrylamide gel electrophoresis and then electroblotted onto nitrocellulose membranes. After re-naturation of the proteins the membranes were incubated with Hfq (150 pmol Hfq as hexamer) as described in Materials and Methods. The CsdAHis₆-Hfq complex was visualized by immuno-detection using anti-Hfq antibodies (lane 1), whereas no CsdA-Hfq complex could be detected with anti-Hfq antibodies after treatment with MN (lane 2). Lane 3, Hfq protein (control) was detected with anti-Hfq antibodies. (B) Co-immunoprecipitation assay with protein CsdA-His6 and Hfq using anti-Hfq antibodies. Lane 1, CsdA-His₆ (10 pmol) was loaded as a positive control for the anti-His antibodies. 25 pmol of purified hexameric Hfq were incubated together with 50 pmol CsdA-His₆ (lane 2) or 50 pmol CsdA-His₆ treated with MN (lane 3) followed by immunoprecipitation using anti-Hfq antibodies bound to the Dynabeads (see Materials and Methods). The co-immunoprecipitated CsdA-His₆ was detected on the western-blot with anti-His antibodies. Only the relevant sections of the immunoblots are shown.