Journal: bioRxiv
Article Title: AGPAT1 is a novel Chikungunya virus receptor on human cells
doi: 10.1101/2025.05.20.655106
Figure Lengend Snippet: (A) Huh7 cells were incubated with CHIKV (MOI 50) on ice for 45 min, followed by CHIKV anti-E2 mouse monoclonal antibody and anti-AGPAT1 rabbit polyclonal antibody for another 30 min. The cells were washed with ice-cold PBS and incubated with anti-mouse Alexa Fluor-647 and anti-rabbit Alexa Fluor-488 antibodies for 30 min. The nuclei were stained with DAPI, and the cells were imaged under the Leica SP8 confocal microscope, as shown in the top left panel (scale bar = 25 µm). The PCC was determined for the co-localization of Alexa Fluor-488 (AGPAT1) and Alexa Fluor-647 (CHIKV) on the plasma membrane. The top right panel shows the line plot showing the co-localization of the red (CHIKV) and green (AGPAT1) fluorescence signals. The bottom panel shows the colocalization of CHIKV and AGPAT1 observed by the super-resolution structured illumination microscopy (SIM) under the Zeiss Elyra PSI microscope (scale bar = 10 µm). The white arrows show the co-localized puncta under 100X magnification and 10X zoom. The representative images are shown. (B) The Huh7 cell lysate was incubated with the purified CHIKV or BSA (negative control). The immunoprecipitation was carried out using the CHIKV anti-E1 antibody. The input cell lysate and the pull-down were Western blotted with AGPAT1 or CHIKV E1 antibodies. A representative blot is shown. (C) Huh7 cells were incubated on ice with rabbit anti-AGPAT1 polyclonal antibody or rabbit IgG (isotype control) for 30 min at 10 µg/ml concentration, followed by incubation with CHIKV (MOI 50) on ice for 30 min. The cells were then incubated with CHIKV anti-E2 mouse monoclonal antibody on ice for 30 min and washed with ice-cold PBS. The cells were stained with Alexa Fluor-647 anti-mouse antibody for 30 min. The nuclei were stained with DAPI, and the cells were imaged using the Leica SP8 confocal microscope (scale bar = 25 µm). The MIFD is shown as mean±SD in the right panel. The experiment was done in triplicate, and a representative image of the stained cells is shown. The Student’s t-test was conducted to analyze the data; *** p <0.001. (D) Huh7 cells were incubated with 5 or 10 mg/ml concentration (indicated in the brackets) of rabbit anti-AGPAT1 antibody or rabbit IgG (isotype control) on ice for 30 min, or without antibody, and then incubated with CHIKV (MOI 1) on ice for 1 h. The cells were then washed with ice-cold PBS and incubated for 3 h at 37 °C. The total RNA from the cells was isolated, and the CHIKV RNA level was determined by qRT-PCR. The CHIKV RNA level in the no-antibody control was taken as 1. The statistical analysis of the data shown as mean±SD was done using the Student’s t-test with Welch’s correction, ** p <0.01.
Article Snippet: The following antibodies were used for the Western blotting: rabbit monoclonal anti-E-Cadherin antibody (CST 3195; Cell Signalling Technologies), rabbit polyclonal GAPDH antibody (GTX100118; GeneTex), rabbit polyclonal anti-Calreticulin antibody (2891; Cell Signalling Technologies), and mouse polyclonal AGPAT1 antibody (ab67018; Abcam).
Techniques: Incubation, Staining, Microscopy, Clinical Proteomics, Membrane, Fluorescence, Purification, Negative Control, Immunoprecipitation, Western Blot, Control, Concentration Assay, Isolation, Quantitative RT-PCR
Proteintech is a verified supplier 
