Subcellular Localization of P-FOXO and P-ERK

Ae. aegypti Aag2 cells were seeded onto coverslips in 12-well plates at a confluency of approximately 1x10⁶ cells/well. Cells were then treated for 24 hours with 1% DMSO, 1 μM DMAQ-B1, 10 μM AKT inhibitor VIII, or combined small molecule treatment supplemented in 2% FBS media as described [14 (link)]. Coverslips were fixed in 4% paraformaldehyde for 10 minutes at room temperature, permeabilized in 0.1% Triton-X-100 for 30 minutes at room temperature and blocked in 1% BSA in TBS for 30 min at 37°C. Primary antibody labeling was completed with anti-P-FOXO (1:100) and anti-P-ERK (1:100) for 2 h at humified room temperature. Secondary antibody labeling was completed using anti-rabbit (Life Technologies A11034) or anti-mouse (Life Technologies A11029) Alexafluor 488 (1:300) by incubating membranes for 1 h at room temperature in the dark. Samples were stained with DAPI (1:100; Cell Signaling 4083), mounted onto coverslips using ProLong Diamond Antifade Mountant (Invitrogen P36961), and imaged using a Leica Sp8X confocal microscope. Localization percentages were determined using Adobe Illustrator 2021 by counting the total number of cells and evaluating if green-fluorescent signal was cytosolic, nuclear, or no signal in relation to DAPI-stained nuclei.