Figure 4
From: Reactivation of latent HIV-1 provirus via targeting protein phosphatase-1
SMAPP1 binds to PP1 and upregulates PP1 activity in vitro. a, b Direct binding of SMAPP1 to recombinant PP1 protein. The direct binding interaction was measured by surface plasmon resonance using a Biacore T-200 instrument. Raw data showing binding of SMAPP1 to PP1 protein are presented in a. The X axis represents time in seconds and the Y axis represents changes in total mass on microchip surface, which was expressed as resonance units. A positive deflection indicated binding of SMAPP1 in solution to PP1 protein that was immobilized on a microchip surface. Each line represents a different concentration of SMAPP1, all between 0 and 50 μM. Each concentration was run three times. In b, the equilibrium dissociation constant (KD) was calculated based on a 1:1 binding model. The KD was calculated as 183 μM for SMAPP1 binding to PP1. Each data point represents the binding level shown in a from different concentrations of SMAPP1 measured just before the end of the injection (~55 s time point). A vertical dashed line represents the small molecule concentration that results in a binding level that is 50% of the extrapolated maximal signal. Experimental data points did not reach to the actual saturation level due to limitation on available SMAPP1 concentration, which resulted in the calculation of KD value from an extrapolated line. c SMAPP1 induces pRb-Tat dephosphorylation by PP1. Recombinant PP1α was assayed with pRb-Tat (75 μM) in the absence or presence of 200 μM SMAPP1 as indicated. The reactions were stopped at indicated time points and the phosphate release was quantified by malachite green assay. Initial velocity was calculated by linear regression. Unpaired t test was used to calculate p value.