FIG 3.

Lengths of 2-5As synthesized by OAS3 and pOAS1 and their capability in activating RNase L. (A) Radiogram of a 20% PAGE showing the lengths of 2-5As synthesized by OAS3 and pOAS1 at various levels of enzymatic activity. Enzymatic activity levels are displayed as synthesized 2-5A in nmol per min for each lane. ATP and 2-5As of various lengths are depicted. The concentration of pOAS1 in the outermost left lane is 48 nM. The concentration of pOAS1, from left to right, is 2.4 nM, 3.6 nM, 4.8 nM, and 6 nM. The concentration of OAS3, from left to right, is 0.8 nM, 1.2 nM, 1.6 nM, and 2 nM. The OAS proteins were activated with 100 μg/ml poly(I·C) for 1 h. (B) Quantification of the 2-5A species synthesized at similar enzymatic activity for OAS3 (0.013 nmol/min) and pOAS1 (0.015 nmol/min). (C) RNase L activity was determined by assaying the integrity of 28S and 18S rRNA. Flp-In T-REx 293 cells stably transfected with hOAS1-FLAG or OAS3-FLAG were either mock transfected or transfected with poly(I·C), and cellular RNA extracts were separated using the Experion HighSens RNA analysis kit. Comparable expression levels of hOAS1-FLAG and OAS3-FLAG were confirmed by Western blot analysis on cell lysates using antibodies against FLAG and GAPDH.