Figure 6. Csd1 expression is diminished when the N-terminal region of CcmA is deleted due to increased Csd7-CcmA interactions that preclude Csd7-Csd1 interactions.
(A) Western blot of whole-cell extracts probed with ɑ-Csd1 polyclonal antibody showing levels of Csd1; ɑ-Cag3 polyclonal antibody was used as a loading control. Strains with ‘csd1c’ indicate an extra copy of csd1 was added at the rdxA locus. Data shown are representative of data from three independent biological replicates. (B) Quantification of Csd1 levels in the ccmA ∆NT csd1c strain relative to the WT csd1c. Relative signal was calculated by dividing Csd1 signal by Cag3 signal for each replicate, then the data was normalized by dividing it by the value from the WT csd1c strain from the same experiment. Data are pooled from three independent biological replicates. Unpaired t-test, **p≤0.001. (C) Co-immunoprecipitation (co-IP) experiments to probe Csd1 levels in relation to Csd7-3x-FLAG-CcmA interactions in H. pylori cells are shown. Top row: Western blot of input fractions probed with ɑ-Cag3 polyclonal antibody as loading control. Second row: Western blot of input fractions probed with ɑ-Csd1 polyclonal antibody. Third row: Western blot of input fractions probed with ɑ-CcmA polyclonal antibody. Fourth row: Western blot probed with ɑ-FLAG monoclonal antibody of IP fractions to detect Csd7-FLAG. Fifth row: Western blot probed with ɑ-Csd1 polyclonal antibody of IP fractions. Bottom row: Western blot probed with ɑ-CcmA polyclonal antibody of IP fractions. Data shown are representative of data from two independent biological replicates. Strains used: LSH100, LSH113, LSH121, DCY26, SSH51A, SSH53A, SSH55A, DCY71, SSH79A, and SSH81B.