Abstract

<p><b>A,</b> HEK293T cells were transfected with plasmids encoding HA-Sumo-1, HA-Sumo-2 and Flag-PM IV together with either empty vector or rat NKAα1. The next day the cells were treated as indicated for 24h, followed by lysis and IP using anti-Flag (PML) antibody. The total lysates before immunoprecipitation and the IP-ed complexes were separated on SDS-PAGE followed by immunoblotting with anti-Flag (PML), anti HA (Sumo-1 and Sumo-2) or anti-ACTIN antibodies. <b>B,</b> Human NKA subunit α1 contains residues Q118 and N129, which are essential for CG binding. Rat NKAα1 lacks these critical residues, rendering it insensitive to CG. HEK293T cells were transfected with either empty vector or rat NKA α1. The following day the cells were treated as indicated for 48h, followed by cell viability determination using Cell Titer Glo assay. Data are means of three replicates and the error bars are standard deviations. <b>C,</b> HEK293T cells were transfected with either NKAα1 or pcDNA. The following day, the cells were recovered and dispensed into 24-well plates and the day after they were pre-treated with 0, 25 nM, 50 nM or 100 nM ouabain for 5h, followed by infection with HSV-1 KOS for 24 hrs. The produced virus was harvested for infection of naïve cells in D, and was detected by immunoblotting with anti-VP16 antibody. <b>D,</b> Phase-contrast image of HEK293T cells 48h after infection with HSV-1 virus produced in cells as described in C. Less virus was produced in cells treated with ouabain, as evident from less VP16 expression (C) and less cell killing (D). The scale in D represents 800 μm. The effect of ouabain was reversed with overexpression of rat NKAα1.</p

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