Cofilin RPPA printing and analyses of cell lysates were provided by Theranostics Health (Gaithersburg, MD, USA). Details of RPPA have been published previously (25). The RPPA directly couples the phospho-cofilin detection antibody with highly sensitive amplification systems that can yield detection sensitivities to fewer than 1000 to 5000 molecules per spot with good linearity (correlation coefficient or R2 = 0.990 to 0.999) and interexperiment precision (R2 = 0.973). Published between-run and within-run analytical precision in our studies was 3 to 13% CV (coefficient of variation) (50). The RPPA technology has been developed and optimized for performance as a fluorescence-based calibrated assay, generally identical in design and analysis to standard ELISA (enzyme-linked immunosorbent assay) or standard clinical immunoassays. Each array consisted of patient cell lysates printed in triplicate two-spot dilutions (neat and 1:4), high and low controls printed in triplicate two-spot dilutions (neat and 1:4), and 6- to 10-point calibrators. The analyte concentration was determined by extrapolation to a nonparametrically determined curve fit of the calibration curve and reported in relative fluorescence units. For data normalization, each protein analyte value (relative p-cofilin value) was normalized to the total amount of protein printed on that spot with a fluorescent stain (Sypro Ruby Blot Stain; Molecular Probes, Eugene, OR) that binds to the amne group of proteins without bias. The protein loading value was also obtained by a calibrated assay technique. This total protein calibrator consisted of a protein lysate with a known concentration, which upon dilution spans the linear dynamic range of protein concentration. Each sample value was then extrapolated to the calibrator. The following were the quantified averages of the total protein levels from each test group: HIV (n = 98), 0.254; HIV + ART (n = 95), 0.259; and HC (n = 100), 0.252. There are no statistically significant differences in the total protein levels between the three groups (HC and HIV, P = 0.77; HC and HIV + ART, P = 0.51; HIV and HIV + ART, P = 0.64).

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