Proteinase K Digestion Assay for FKJcR2a

The proteinase K digestion protection assay was per- formed as described in [74 (link)] with some modifications. Briefly, wild-type or the E530A variant of FK- JcR2a RNA was electroporated into Huh7.5 cells. The cells were collected at 48 h pe in PBS, subsequently resuspended in proteinase K buffer (50mM Tris-Cl, pH 8.0, 10 mM CaCl₂, 1 mM DTT) and subjected to 5 freeze-thaw cycles. Cell lysates were obtained after centrifugation, divided into three groups: untreated or treated with 50 μg/μl of proteinase K (Roche, Mannheim, Germany) on ice for 1 h, and pretreated with 5% (vol/vol) Triton X-100 before proteinase K treatment on ice for 1 h. proteinase K was then inactivated with 5 mM phenylmethylsulfonyl fluoride (PMSF) on ice for 10 min and cell lysates were mixed with sample buffer. The amount of core protein was determined by Western blot.