Fig. S2.

Expression of a putative truncated Mili isoform in the brain. (A) Expression of Piwil2-like protein 60 (PL2L60), a Mili variant in the adult testes. Five micrograms of testes whole cell lysates from 8-wk-old WT (+/+) or Mili^−/− (−/−) mice were run on a SDS/PAGE followed by Western blotting using a C-terminal (601–680) and N-terminal (107–121) anti-Mili antibodies. Note that the 60- and 80-kDa species representing PL2L60 and a putative PL2L80 proteins were only detected in WT lysates when blotted with the C-terminal anti-Mili, confirming that these were N-terminally truncated products of Mili as previously described (29). The exact mechanism for the generation of PL2L60 is not clear. In the Mili^−/− mice, the Mili gene was disrupted by homologous recombination within exon 2–5 (22), thus leading to the expression of exon 6–23 and a low level expression of exon 1–7 (29). Note that PL2L60 described as the major variant was only depleted but not completely absent from Mili^−/− lysates due to transcription from a distal promoter located 3′ of the disrupted region (intron 10, a region still intact in Mili^−/− mice) of the Mili gene and residual transcription at the 5′, thus encoding some PL2L60 protein with N-terminal truncation (encoding part of the N-terminal region and the PAZ domain) but with intact C-terminally located MID and PIWI domains (29). (B) Expression of PL2L60 in the brain. Cytoplasmic and nuclear fractions of whole 8-wk-old WT and Mili^−/− brains were Western blotted using a C-terminal anti-Mili antibody. The band corresponding to PL2L60 was absent when reblotted with an N-terminal anti-Mili antibody, suggesting that PL2L60 could be a N-terminally truncated product of Mili. MAP2c and NeuN were respectively used as markers for cytoplasmic and nuclear fractions. Experiment was repeated two to three times.