Figure 6. Sam68 enhances translation of... | Rockefeller University Press

Figure 6.

$Figure 6. Sam68 enhances translation of Spag16, Nedd1, and Spdya mRNAs in germ cells. (A) RT-PCR analysis of the distribution of Spag16, Nedd1, Spdya, or Gapdh mRNAs in the fractions of the sucrose gradients derived from sucrose gradient fractionation of germ cell extracts from Sam68+/− or Sam68−/− mice. Densitometric analysis of the signal in each fraction was performed, and the results were represented as the percentage of total signal in all fractions in the bottom panels. (B) Quantitative real-time PCR analysis of Spag16, Nedd1, Spdya, Tpn2, and Prm2 mRNA levels in the polysomal (1–5) and RNP (8–10) fractions. Gapdh mRNA was used as an internal standard for the real-time PCR analysis. (C) Western blot analysis of Sam68, SPAG16, NEDD1, and SPDYA proteins in germ cells isolated from Sam68+/+ or Sam68−/− testes. ERK2 was used as a loading control. (D) Immunofluorescence analysis of SPAG16 and NEDD1 proteins in germ cells isolated from Sam68+/+ or Sam68−/− testes. Hoechst costaining was performed to distinguish pachytene spermatocytes (Pc) from round spermatids (Spt). Bars, 10 µm.$

Sam68 enhances translation of Spag16, Nedd1, and Spdya mRNAs in germ cells. (A) RT-PCR analysis of the distribution of Spag16, Nedd1, Spdya, or Gapdh mRNAs in the fractions of the sucrose gradients derived from sucrose gradient fractionation of germ cell extracts from Sam68^+/− or Sam68^−/− mice. Densitometric analysis of the signal in each fraction was performed, and the results were represented as the percentage of total signal in all fractions in the bottom panels. (B) Quantitative real-time PCR analysis of Spag16, Nedd1, Spdya, Tpn2, and Prm2 mRNA levels in the polysomal (1–5) and RNP (8–10) fractions. Gapdh mRNA was used as an internal standard for the real-time PCR analysis. (C) Western blot analysis of Sam68, SPAG16, NEDD1, and SPDYA proteins in germ cells isolated from Sam68^+/+ or Sam68^−/− testes. ERK2 was used as a loading control. (D) Immunofluorescence analysis of SPAG16 and NEDD1 proteins in germ cells isolated from Sam68^+/+ or Sam68^−/− testes. Hoechst costaining was performed to distinguish pachytene spermatocytes (Pc) from round spermatids (Spt). Bars, 10 µm.