Figure 5. Cytoplasmic Sam68 associates... | Rockefeller University Press

Figure 5.

$Figure 5. Cytoplasmic Sam68 associates with polyadenylated mRNAs and the translational machinery in germ cells. (A and B) Immunofluorescence analysis of germ cell populations enriched in secondary spermatocytes (A) or round spermatids (B) demonstrates that Sam68 is frequently localized in the cytoplasm in these cells. Representative examples of cytoplasmic localization are indicated by arrows. Bars, 10 µm. (C and D) Pull-down assay using the 5′-cap analogue m7GTP-Sepharose beads (M7GTP) to isolate the translation initiation complex from male germ cells (C) or HEK293 cells (D). Control pull-down assays were performed using Sepharose beads (sepharose). The panels show Western blot analysis of the Sam68, eIF4G, and eIF4E proteins in the input and bound in the pull-down assays using pachytene spermatocyte (s.cytes) extracts or extracts from a population enriched in secondary spermatocytes (II s.cytes) (C). Bottom panels show densitometric analysis of the pull-down assays. Data are represented as ratios between Sam68 and eIF4E or eIF4G and eIF4E band intensities. (E) Absorbance profile (OD = 254 nm) of sucrose gradient sedimentation of cell extracts from a population enriched in secondary spermatocytes. Extracts were either untreated (control) or treated with EDTA. The gradients were collected in 10 fractions, starting from the bottom heavy fractions, and the bottom panels show the Western blot analysis of Sam68 and PABP1 distribution in each fraction of the gradient. (F) RNP capture assay using oligo (dT)–cellulose beads in the presence or absence of excess free polyA synthetic RNA and polysomal (1–5) fractions derived from sucrose gradient fractionation of cell extracts as described in E. The input loaded in the pull-down experiment and the proteins bound to the beads were separated by SDS-PAGE and immunoblotted with the anti-Sam68 antibody. (G) Western blot analysis of the coimmunoprecipitation between Sam68 and PABP1 from germ cell extracts. Samples were immunoprecipitated with either nonimmune (Ctrl) or anti-Sam68 (α-Sam68) IgGs. Immunoprecipitated proteins were separated on 10% SDS-PAGE and immunoblotted with anti-Sam68 or anti-PABP1 antibodies.$

Cytoplasmic Sam68 associates with polyadenylated mRNAs and the translational machinery in germ cells. (A and B) Immunofluorescence analysis of germ cell populations enriched in secondary spermatocytes (A) or round spermatids (B) demonstrates that Sam68 is frequently localized in the cytoplasm in these cells. Representative examples of cytoplasmic localization are indicated by arrows. Bars, 10 µm. (C and D) Pull-down assay using the 5′-cap analogue m7GTP-Sepharose beads (M7GTP) to isolate the translation initiation complex from male germ cells (C) or HEK293 cells (D). Control pull-down assays were performed using Sepharose beads (sepharose). The panels show Western blot analysis of the Sam68, eIF4G, and eIF4E proteins in the input and bound in the pull-down assays using pachytene spermatocyte (s.cytes) extracts or extracts from a population enriched in secondary spermatocytes (II s.cytes) (C). Bottom panels show densitometric analysis of the pull-down assays. Data are represented as ratios between Sam68 and eIF4E or eIF4G and eIF4E band intensities. (E) Absorbance profile (OD = 254 nm) of sucrose gradient sedimentation of cell extracts from a population enriched in secondary spermatocytes. Extracts were either untreated (control) or treated with EDTA. The gradients were collected in 10 fractions, starting from the bottom heavy fractions, and the bottom panels show the Western blot analysis of Sam68 and PABP1 distribution in each fraction of the gradient. (F) RNP capture assay using oligo (dT)–cellulose beads in the presence or absence of excess free polyA synthetic RNA and polysomal (1–5) fractions derived from sucrose gradient fractionation of cell extracts as described in E. The input loaded in the pull-down experiment and the proteins bound to the beads were separated by SDS-PAGE and immunoblotted with the anti-Sam68 antibody. (G) Western blot analysis of the coimmunoprecipitation between Sam68 and PABP1 from germ cell extracts. Samples were immunoprecipitated with either nonimmune (Ctrl) or anti-Sam68 (α-Sam68) IgGs. Immunoprecipitated proteins were separated on 10% SDS-PAGE and immunoblotted with anti-Sam68 or anti-PABP1 antibodies.

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