Figure 7.

Piwi and Aub, but Not AGO3 Can Be Loaded by the Somatic Primary piRNA Biogenesis Pathway

(A) Shown are IF images of OSCs transfected with the indicated GFP-tagged PIWI proteins (green) stained for Armi (blue) and Krimp (red). The bottom row shows the overlay of all channels.

(B) Shown are relative levels of indicated FLAG/HA-PIWI mRNAs isolated from transfected cells (n = 3; error bars indicate StDev).

(C) Shown are protein levels of FLAG/HA-PIWI proteins isolated from transfected cells in comparison to Lamin levels.

(D) IP-western analysis indicating efficient immunoprecipitation of tagged Piwi and Aub, but not AGO3 from lysate of transfected OSCs.

(E) CIP-Kinase experiment indicating efficient loading of tranfected Piwi and Aub in OSCs.

(F) Western blot indicating that ectopically expressed GFP-Aub is destabilized in OSCs upon knockdown of piRNA biogenesis factors.

(G) Annotation of small RNAs isolated from indicated IPs from OSCs.

(H) Size profile of small RNAs isolated from indicated IPs.

(I) First nucleotide bias of small RNAs isolated from indicated IPs.

(J) Scatter plot of antisense piRNAs isolated from indicated IPs mapping to Repbase TEs.

(K) Profiles of small RNAs isolated from indicated IPs mapping uniquely to the flamenco cluster (left; 200 nt walking window) to the ZAM TE (middle) or to the traffic jam locus (right). All plots were normalized to one million small RNAs in the respective library.