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. 2022 Sep 8;11:e80111. doi: 10.7554/eLife.80111

Figure 5. The bactofilin domain interacts with Csd5 and Csd7, and the N-terminal region inhibits Csd7 binding to CcmA.

(A, B) Co-immunoprecipitation (co-IP) experiments to probe Csd5-2x-FLAG-CcmA interactions (A) and Csd7-3x-FLAG-CcmA interactions (B) in H. pylori cells are shown. Top row: Ponceau S staining of input fractions. Second row: Western blot probed with ɑ-CcmA polyclonal antibody of input fractions. Third row: Western blot probed with ɑ-FLAG monoclonal antibody of IP fractions. Bottom row: Western blot probed with ɑ-CcmA polyclonal antibody of IP fractions. (C) Co-IP experiments to probe Csd7-3x-FLAG interactions with CcmA and Csd5 in H. pylori strains with and without CcmA. Top row: Ponceau S staining of input fractions. Second row: Western blot probed with ɑ-SH3 polyclonal antibody of input fractions to detect the SH3 domain of Csd5. Third row: Western blot probed with ɑ-CcmA polyclonal antibody of input fractions. Fourth row: Western blot probed with ɑ-FLAG monoclonal antibody of IP fractions to detect Csd7-3x-FLAG. Fifth row: Western blot probed with ɑ-SH3 polyclonal antibody to detect the SH3 domain of Csd5 in IP fractions. Bottom row: Western blot probed with ɑ-CcmA polyclonal antibody of IP fractions. Data shown are representative of data from three independent biological replicates. Strains used: KHB157, LSH100, SSH59A, SSH58A, SSH68A, SSH60A, SSH65A, DCY71, DCY77, SSH79A, SSH78A, SSH82, SSH80A, and SSH81B.

Figure 5—source data 1. Raw, unedited Western blots probed with ɑ-CcmA and ɑ-FLAG antibodies to detect CcmA and Csd5-FLAG or Csd7-FLAG in co-immunoprecipitation experiments.

Figure 5.

Figure 5—figure supplement 1. The C-terminus of CcmA-HaloTag is not required for interactions with Csd5.

Figure 5—figure supplement 1.

ɑ-FLAG immunoprecipitation of strains expressing Csd5-FLAG and WT CcmA-HaloTag, CcmA ∆CT-HaloTag, or CcmA BD only –HaloTag. Top row: Ponceau S staining of input fractions. Second row: Western blot of inputs probed with ɑ-HaloTag monoclonal antibody to detect CcmA-HaloTag, both top and second row are images of the same membrane. Third row: Western blot of IP fractions probed with ɑ-FLAG monoclonal antibody to detect Csd5-FLAG. Bottom row: Western blot of IP fractions probed with ɑ-HaloTag monoclonal antibody to detect CcmA-HaloTag. Strains used: SSH87A, SSH89A, and SSH97A.
Figure 5—figure supplement 1—source data 1. Raw, unedited Western blots probed with ɑ-HaloTag and ɑ-FLAG antibodies to detect CcmA-HaloTag and Csd5-FLAG in co-immunoprecipitation experiments.