Kit e1501

NH1 and NH2 cells grown in the logarithmic phase were seeded into 24-well plates for 12 h and then exposed to dimeththyl sulfoxide (DMSO) or different concentrations of compound 7 for 12 h. Then, cell lysates were prepared and measured using kit E1501 from Promega according to the manufacturer’s instructions, and the numbers of cells were normalized based on their contained α-tubulin levels.

For the luciferase assay, the HeLa-based NH1 cells [19 (link), 26 (link)] containing an integrated HIV-1 LTR-luciferase reporter construct but expressing no Tat, as well as its derivative NH2 cells [26 (link)], which also harbor an integrated Tat-HA expression vector, were used. Cells were then treated with different concentrations of extracts for 24 h and subjected to the luciferase assay using kit E1501 from Promega and following the manufacturer's instructions. Lysates were prepared from an approximately equal number of cells and normalized based on their contained α-tubulin levels among all the samples.

The HeLa-based NH1 cell line containing an integrated HIV-1 LTR-luciferase reporter plasmid (He et al., 2010 (link)) was transfected with constructs expressing Tat(C22G) only or together with the indicated AFF1 proteins. AFF1 mutants were chosen based on their surface exposed position on the second αhelix in the homologous AFF4 (Schulze-Gahmen et al., 2014 (link)), with the exception of Val 67, which is only partially exposed. At 48 hr post transfection, total cell lysates were prepared and luciferase activity was measured with kit E1501 from Promega (Madison, WI, USA). An aliquot of cell lysates was analyzed by western blotting with the indicated antibodies to detect the level of the transfected AFF1 protein and the endogenous α-Tubulin.