FIGURE 3.

Prmt5 exists only in complex with Mep50 in vivo and is found in the nucleus and on chromatin. a, total clarified egg extract was applied to a Superose 6 sizing column, and every other fraction was immunoblotted for Prmt5 and Mep50. Elution positions of 670 and 158 kDa mass markers are shown above. b, endogenous co-immunoprecipitation of Prmt5 with α-Mep50 in egg extract. c, 0.5 μl of clarified egg extract and 0.5 μl of NPE were immunoblotted for Prmt5 and PCNA (a nuclear marker). A small proportion of Prmt5 was found in the nuclear extract. d, sperm pronuclei were assembled in egg extract and collected immediately after incubation (1 min; first lane) or after 45 min (second lane). Chromatin was isolated through a sucrose cushion and immunoblotted for Prmt5, H2A, H3, and Mcm7. e, rPrmt5-Mep50, total egg extract, XL2 whole cell extract, and A6 whole cell extract were immunoblotted for Prmt5, Mep50, and PCNA. The bottom panel shows the total protein content in each lane by Direct Blue 71 staining of the membrane. The protein contents for the egg and whole cell extracts were normalized by a Bradford assay, with an identical total mass of protein loaded in each lane. r, recombinant. f, purified histones from Xenopus eggs, XL2 cells, and A6 cells were immunoblotted for H2A (as control) and R3me1 (Proteintech). The antibodies recognize both H2A and H2A.X-F in the egg extract; for the R3me1 antibody, we refer to both as H2A/H2A.X-F because we cannot distinguish between H2A and its variant in this case. Equal masses of histones were loaded in each lane as shown by Coomassie staining (bottom). IP, immunoprecipitation.