FIGURE 7.

(A) Western analysis of 5 h 10%–30% glycerol-gradient fractions 5–13 of lysates from PF CN cells with myc-tagged endogenous KREN1, KREN2, or KREN3, and in which the tet-regulatable WT KREPB4 allele was expressed (E) or repressed (R) for 4 d. Western blots were probed with monoclonal antibodies against the myc-epitiope tag to detect the endonucleases, and against editosome proteins KREPA1, KREPA2, KREL1, and KREPA3. (B) Western analysis of input lysates for glycerol-gradient fractionation described in A. (C) anti-KREPA2 IP of editosomes from pooled ∼20S glycerol-gradient fractions 8–11 shown in A. Immunoprecipitates and input pooled fractions were probed with antibodies as in A. Asterisk indicates the heavy chain of the IP antibody. (D) Anti-KREPA2 IP and (E) anti-V5 IP of editosomes from total lysates of PF CN cells with myc-tagged endogenous KREN1, KREN2, or KREN. Cells were either not expressing KREPB4 (CN), or were exclusively expressing V5-tagged WT, G163V, or G163R KREPB4 alleles. Immunoprecipitates and input lysates were probed with antibodies as in A and with anti-V5-tag antibody to detect the V5-tagged WT or mutant KREPB4 proteins.