Figure 5.

Farnesylation of J3 affects membrane association of AGO1. A–D, Western blotting of AGO1, J2/J3, HSP70, and HSP90 in total and microsome fractions prepared from inflorescence lysates of the indicated genotypes. For total fractions, equal loading was verified by Coomassie staining (CBB); for microsome fractions, Western blots were probed with SIP2 antibodies. In A–C, different sections of the membranes used for analysis of total lysates and microsome fractions were used for verification of protein loading. In these cases, the leftmost molecular weight standard refers to the membrane used for total lysates, whereas the rightmost molecular weight standard refers to the membrane used for microsome fractions. E, RNase sensitivity of membrane-bound AGO1. Western blotting showing AGO1 abundance in microsome fractions with or without treatment with RNase A (10 μg/ml). Top, RNase A was added to hypertonic lysis buffer and was present throughout microsomal fractionation. Bottom, microsomes were prepared with RNase-free buffer, but were resuspended and washed for 15 min in lysis buffer with or without 10 μg/ml of RNase A.