Ago2 forms HMW RISC with GW182 in activated CD8+ T cells. (A) Schematic of the experimental protocol indicating that T cells grow upon activation and develop into mature d6 cytolytic T cells capable of secreting effector molecules. Cytotoxic effector molecules are packaged in the endosome in the cytoplasm and then secreted, as indicated by the dense blue vesicles. (B) Ago2 was immunoprecipitated from cell lysates (each lane representing protein from 2 × 107 cells), collected from a time-course of CD8+ T-cell activation, and western blots were probed with Ago2 and GW182 antibodies. (C) Quantification of the western blots measured on a LI-COR imager as GW182 band intensity relative to Ago2 band intensity, shown normalized to expression of Ago2 on d2. Mean and range from two independent experiments. (D) Western blot from Ago2 and GW182 IPs showing coimmunoprecipitated (bound) and residual protein (unbound) fractions, from naïve d0 and d6 activated CD8+ T-cell lysates (2 × 107 cells per IP), probed with Ago2 and GW182 antibodies, as indicated. (E,F) Size exclusion chromatography fractions from naïve d0 (E) and d2 activated (F) CD8+ T-cell lysates. A total of 0.9 mg protein lysate (from 9 × 107 cells) was added for (E) and 1.6 mg (from 8 × 107 cells) for (F). Protein elution curves show fraction volume (in mL) and absorption at 280 nm (in milli-Absorption Units) with location in elution profile of protein size standards noted. Western blot from precipitated protein from fractions probed with Ago2 antibody. (G) Ago2 IP from HMW, intermediate and LMW fractions pooled together as indicated on western blots and elution curves (d2). Western blot of IP samples probed with Ago2 and GW182 antibodies.
