Journal: Frontiers in Microbiology

Article Title: Isobaric tags for relative and absolute quantification-based proteomic analysis of host-pathogen protein interactions in the midgut of Aedes albopictus during dengue virus infection

doi: 10.3389/fmicb.2022.990978

Figure Lengend Snippet: PC protein inhibits the expression of DENV-E protein. (A) Empty vector were transfected into C6/36 cells, and cells were inoculated with DENV (MOI = 1) 48 h after transfection, lysates of C6/36 cells at 24 hpi were detected by SDS-PAGE and WB using the antibodies as indicated. (B,C) 0, 1, 2 μgpAC5.1b-myc-YFP-Cuticle-HA was transfected into C6/36 cells, respectively, and DENV (MOI = 1) was infected 48 h after transfection. The cells’ RNA and protein were extracted from the cells at 24 hpi and detected by qRT-PCR and WB as indicated, respectively. (D) BHK-21 cells were transfected with pcDNA3.1(+)-YFP-Cuticle-HA plasmids and infected with DENV (MOI = 1) at 24 h after transfection. Cells were fixed at 24 hpi, and incubated with a DENV-E protein monoclonal antibody and the corresponding fluorescent secondary antibody. A YFP-PC-HA fusion protein was expressed to detect PC protein (green), localization of DENV-E was detected with antibodies to fluorescent secondary antibody (red), nuclei were stained with DAPI (blue). The upper and lower images in each column are of two fields of view in the same slide. (A,E) COS7 cells were transfected with pcDNA3.1 (+)-YFP-EEV6-HA plasmid and infected with DENV (MOI = 1) at 24 h after transfection, and proteins were extracted at 24 hpi for WB and Co-ip analysis. All experiments were performed at least three times, *denotes a P- value < 0.005, **denotes a P- value < 0.001.

Article Snippet: The primary antibodies comprised a mouse anti-DENV-E monoclonal antibody (1:5,000; Invitrogen, Waltham, MA, United States), a mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:2,000; PTM Biolabs, Hangzhou, China), and a rabbit anti-HA monoclonal antibody (1:2,000; PTM Biolabs).

Techniques: Expressing, Plasmid Preparation, Transfection, SDS Page, Infection, Quantitative RT-PCR, Incubation, Staining, Co-Immunoprecipitation Assay