Journal: Structure (London, England : 1993)

Article Title: Structural basis of pseudoGTPase-mediated protein-protein interactions

doi: 10.1016/j.str.2025.07.009

Figure Lengend Snippet: (A) Diagrams of HA-tagged full-length AP1 σ subunit and 3xFLAG-tagged WT or mutant AAGAB used in immunoprecipitation (IP) experiments. (B) Representative immunoblots showing the interaction of 3xFLAG-tagged WT or mutant AAGAB with HA-tagged AP1 σ subunit. The 3xFLAG-AAGAB proteins were transiently expressed in AAGAB KO HeLa cells with an empty vector (control) or plasmids encoding the HA-tagged AP1 σ subunit. The 3xFLAG-AAGAB proteins were immunoprecipitated from cell lysates using anti-FLAG antibodies, and the presence of 3xFLAG-AAGAB and HA-tagged AP1 σ subunit in the immunoprecipitants was detected using anti-FLAG and anti-HA antibodies, respectively. (C) Representative confocal microscopy images from three experiments showing surface levels of TfR in AAGAB KO HeLa cells expressing WT or mutant AAGAB. Surface TfR levels of non-permeabilized cells were labeled using anti-TfR antibodies and Alexa Fluor 568-conjugated secondary antibodies (red). Nuclei were stained with Hoechst 33342 (blue). Scale bars: 20 μm. (D) Flow cytometry measurements of the surface levels of TfR in AAGAB KO HeLa cells expressing the indicated proteins. Data are presented as mean ± s.d., n = 3. *** p < 0.001. (E) Representative immunoblots showing the expression of the indicated proteins in AAGAB KO HeLa cell expressing WT or mutant AAGAB. (F) The relative expression levels of indicated proteins were normalized to WT AAGAB samples. Data are presented as mean ± s.d., n = 3. *** p < 0.001, ** p < 0.01, * p < 0.05, n.s., p > 0.05. In (D) and (F), p values were calculated using one-way ANOVA and Dunnett’s multiple comparison test. Mock: cells transfected with an empty vector. See also .

Article Snippet: Surface TfR was stained using monoclonal anti-TfR antibodies (DSHB, #G1/221/12) at a final concentration of 0.1 μg/mL and Alexa Fluor 568-conjugated secondary antibodies (Thermo Fisher Scientific, #A11004) at a final concentration of 1 μg/mL.

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Plasmid Preparation, Control, Confocal Microscopy, Expressing, Labeling, Staining, Flow Cytometry, Comparison, Transfection