Journal: Frontiers in Immunology

Article Title: Multifunctional regulation of VAMP3 in exocytic and endocytic pathways of RBL-2H3 cells

doi: 10.3389/fimmu.2022.885868

Figure Lengend Snippet: No FcϵRI upregulation was observed in cell surface expression or internalization The expression of each subunit of FcϵRI in VAMP3 KD cells was determined. (A) Gene expression was analyzed using quantitative RT-PCR. (B) Protein expression was analyzed using western blot analysis. (C) Cell surface expression levels of FcϵRIα that binds to the IgE Fc region were analyzed using flow cytometry in the cells at steady state. (D) Ag-induced time-dependent reduction in cell surface FcϵRI-bound IgE was analyzed using flow cytometry. A representative blot is shown for three independent experiments, and the band density normalized to that of unstimulated cells is shown below each band (B) . Data are presented from three independent experiments. Three biological repeats were quantified in a single run of PCR in the qPCR analysis. NT: control knockdown with a non-targeting shRNA plasmid. Ag: antigen. gMFI: geometric mean fluorescence intensity. *p<0.05, ***p<0.001; ns, not significant.

Article Snippet: For the immunoprecipitation of FcϵRI, supernatants were incubated with goat anti-mouse IgE (Southern Biotech, Birmingham, AL, USA) for 1 h at 4°C followed by incubation with protein A-Sepharose beads (GE Healthcare, Chicago, IL, USA) for 1 h at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, shRNA, Plasmid Preparation, Fluorescence