Figure 4. Disrupting psGD:σ interaction reduces σ subunit level in cells and compromises membrane trafficking.

(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). Images were captured using a 100× oil immersion objective on a Nikon A1 Laser Scanning confocal microscope. Scale bars: 20 μm. (d) Flow cytometry measurements of the surface levels of TfR in AAGAB KO HeLa cells expressing the indicated proteins. Cells were disassociated by Accutase and stained with monoclonal anti-TfR antibodies and APC-conjugated secondary antibodies. APC fluorescence measurements of ~5000 cells were collected on a CyAn ADP analyzer. Mean APC fluorescence was normalized to the control sample in which an empty vector was transfected into AAGAB KO cells. Data are presented as mean±s.d., n=3. ***P<0.001. P values were calculated using one-way ANOVA and Dunnett’s multiple comparison test. (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. P values were calculated using one-way ANOVA and Dunnett’s multiple comparison test. The representative immunoblots are shown in (e). Mock: cells transfected with an empty vector.