Over expression of ANKRD22

To define the impact of ANKRD22 protein on HIV Infection. HEK293T cells were reverse transfected with plasmid encoding each gene. Briefly, 10 μg of pCMV6 encoding ANKRD22 gene (OriGene, Rockville, MD, USA) was incubated with 30 μL of TransIT-293 in 600 uL of OPT-MEM for 30 minutes at RT, and then cultured with 6x10⁶ 293T cells in 10 mL of D10 media in a 100 mm tissue culture dish (Becton Dickinson, Franklin Lakes, NJ, USA) for 2 days. As a control, empty pCMV6 vector (OriGene) was transfected into the cells. The transfected cells were collected and then expression of ANKRD22 was confirmed by WB. HIV infection was conducted using 2 x10⁶ cells in 500 uL of D10 media. The cells were infected with 100 ng p24 /mL of HIVLuc-VSVG for 2 hours at 37°C on the rotator. After infection, the cells were washed with D10 and then cultured at 50x10³ cells/ 96 well plate for 2-days in octuplicate in 200 μL D10 media. The cells were lysed using Bright-Glo (Promega) and Luciferase expression was measured using the Enspire Multimode Plate Reader.