FIG. 1.

Expression of Bclw mRNA and protein in male mouse germ cells. A) RNA was extracted from preparations of enriched germ cells, from testes of heterozygous or homozygous mutant atrichosis (at) mice, from testes of normal (X/Y) or sex-reversed mice (XX Sxr), or from testes of mice at Postnatal Day 7, 12, 17, 22, and 27 during prepubertal development and from adults. The percentage of contaminants in the enriched germ cell preparations is as described in Materials and Methods. Testes from at/at and XX Sxr animals contain extremely few or no germ cells, respectively; thus, any signal is derived from somatic cells of the testis. The RNAs were subjected to Northern blot analysis using a full-length Bclw cDNA probe. To determine whether equal amounts of total RNA had been loaded, the gel was stained with ethidium bromide (EtBr), photographed under ultraviolet (UV) light before transfer, and the blots hybridized with an actin cDNA (lower and middle panels, respectively). A, Testes from adult mouse; RB, residual bodies; RS, round spermatids; at/+, testis from heterozygote atrichosis mouse; at/at, testis from homozygote atrichosis mouse; X/Y, testis from wild-type mouse; XX Sxr, testis from sex-reversed mouse; 7, 12, 17, 22, and 27, testes from Postnatal Day 7, 12, 17, 22, and 27 mice. B) Western blot analysis of total protein in preparations of enriched germ cells. Cell preparations enriched for round spermatids (RS) or residual bodies (RB) from adult animals were solubilized, and total protein was analyzed by Western blot analysis using affinity-purified rabbit antisera against a peptide sequence from mouse Bclw [5]. The positive and negative controls include extracts of COS cells transfected with either a mammalian expression vector with a mouse Bclw cDNA insert (COS + Bclw) or the same vector without a cDNA insert (COS). Arrowheads indicate the position of the Bclw-specific band. Note the absence of Bclw protein in both RS and RB. C) To determine whether equal amounts of protein had been loaded, the blot shown in B was stained with Ponceau S before reacting with antisera. Arrowheads denote the approximate size of Bclw (22 kDa). c, COS cells; cb, COS cells transfected with vector expressing Bclw; m, molecular weight markers; rb, enriched for residual bodies; rs, enriched for round spermatids.