Journal: iScience

Article Title: Legionella employs the multimodal ubiquitination of Sec22b to modulate SNARE pairing

doi: 10.1016/j.isci.2025.114341

Figure Lengend Snippet: Ubiquitination of Sec22b promotes non-canonical SNARE pairings with Stx3 and Stx4 (A) HEK293T-FcγRII cells stably expressing FLAG-Sec22b were infected with the indicated L. pneumophila strains at an MOI of 80 for 1 h. FLAG-Sec22b derivatives were immunoprecipitated from the cell lysates at the indicated time points using anti-FLAG beads. The cell lysates (input) and the immunoprecipitated proteins (IP: FLAG) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The intensity values of Stx3 protein bands were quantified using the ChemiDoc System with Image Lab software (Bio-Rad), normalized to the value of the no-infection condition (no bacteria), and displayed below the immunoblotting data. (B) HEK293T-FcγRII cells stably expressing FLAG-Sec22b S137A were infected with the indicated L. pneumophila strains at an MOI of 80 for 1 h, and analyzed as described in (A). The intensity values of Stx3 protein bands were quantified as described in (A) and displayed below the immunoblotting data. (C) HEK293T-FcγRII cells stably expressing FLAG-Sec22b or FLAG-Sec22b S137A were infected with the wild-type strain Lp01 at an MOI of 80 for 1 h, and analyzed as described in (A). (D) Intensity values of Stx3, Stx4, and Stx18 protein bands from (C) quantified as described in (A), normalized to the respective Syntaxin values bound to Sec22b WT. Values represent the mean ± SEM from three independent experiments. ns, not significant. ∗ p < 0.05, ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-Stx3 rabbit polyclonal antibody (15556-1-AP) and anti-Stx4 rabbit monoclonal antibody (14988-1-AP) were purchased from Proteintech.

Techniques: Ubiquitin Proteomics, Stable Transfection, Expressing, Infection, Immunoprecipitation, SDS Page, Western Blot, Software, Bacteria

Journal: iScience

Article Title: Legionella employs the multimodal ubiquitination of Sec22b to modulate SNARE pairing

doi: 10.1016/j.isci.2025.114341

Figure Lengend Snippet: Ubiquitination of Sec22b promotes non-canonical SNARE pairings with Stx3 and Stx4 (A) HEK293T-FcγRII cells stably expressing FLAG-Sec22b were infected with the indicated L. pneumophila strains at an MOI of 80 for 1 h. FLAG-Sec22b derivatives were immunoprecipitated from the cell lysates at the indicated time points using anti-FLAG beads. The cell lysates (input) and the immunoprecipitated proteins (IP: FLAG) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The intensity values of Stx3 protein bands were quantified using the ChemiDoc System with Image Lab software (Bio-Rad), normalized to the value of the no-infection condition (no bacteria), and displayed below the immunoblotting data. (B) HEK293T-FcγRII cells stably expressing FLAG-Sec22b S137A were infected with the indicated L. pneumophila strains at an MOI of 80 for 1 h, and analyzed as described in (A). The intensity values of Stx3 protein bands were quantified as described in (A) and displayed below the immunoblotting data. (C) HEK293T-FcγRII cells stably expressing FLAG-Sec22b or FLAG-Sec22b S137A were infected with the wild-type strain Lp01 at an MOI of 80 for 1 h, and analyzed as described in (A). (D) Intensity values of Stx3, Stx4, and Stx18 protein bands from (C) quantified as described in (A), normalized to the respective Syntaxin values bound to Sec22b WT. Values represent the mean ± SEM from three independent experiments. ns, not significant. ∗ p < 0.05, ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-Stx3 rabbit polyclonal antibody (15556-1-AP) and anti-Stx4 rabbit monoclonal antibody (14988-1-AP) were purchased from Proteintech.

Techniques: Ubiquitin Proteomics, Stable Transfection, Expressing, Infection, Immunoprecipitation, SDS Page, Western Blot, Software, Bacteria