Journal: Nucleic Acids Research

Article Title: Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification

doi: 10.1093/nar/gkad545

Figure Lengend Snippet: Immunoprecipitation of endogenous translating ribosomes mediated by the C-terminal IDRs of the P-stalk. ( A ) A structural model of human 80S ribosome containing ribosomal P-stalk generated from three coordinates [Protein data bank code: 3A1Y, 4BEH and 6EK0 ( , , )]. The C-terminal IDR of P0 is modeled arbitrarily. The epitopes of the anti-P0 antibody (9D5) are shown as a stick model. Colors are as follows: 40S ribosomal subunit (cyan), 60S ribosomal subunit (pale yellow), and ribosomal protein P0 (orange). ( B ) Immunoprecipitation of the endogenous ribosome and ribosome-bound RNA with the anti-ribosomal protein P0 antibody (9D5) or isotype control IgG (NC) from the cytosolic lysate of HEK293 cell in the presence or absence of RNase A, followed by western blotting. The input contained 1% of the lysate used for immunoprecipitation. Asterisks (*) indicate non-specific signals from the antibodies (9D5 or NC). ( C ) Examples of capillary electrophoresis profiles for cytosolic RNAs and immunoprecipitated RNAs from HEK293 cells. Two independent experiments were performed for each condition (#1, #2). The lower marker (LM) indicates internal standards (25 nt). RNA ladder (the Agilent RNA 6000 Pico kit) was used as a size marker.

Article Snippet: The isotype control antibody for 9D5 (mouse IgG2a κ) was purchased from Proteintech (65208-1-IG).

Techniques: Immunoprecipitation, Generated, Control, Western Blot, Electrophoresis, Marker

Journal: Nucleic Acids Research

Article Title: Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification

doi: 10.1093/nar/gkad545

Figure Lengend Snippet: Immunoprecipitation of the endogenous ribosome from diverse eukaryotes mediated by the C-terminal IDRs of the P-stalk. ( A ) Sequence alignment of the C-terminal 12 amino acids of ribosomal protein P0 in diverse eukaryotes. The 9D5 anti-P0 antibody recognizes these sequences. (B–D) Immunoprecipitation of the endogenous ribosome with the 9D5 antibody or isotype control IgG (NC) from whole zebrafish embryo ( B ), Drosophila S2 cells ( C ) and adult C. elegans ( D ) in the presence of RNase A, followed by western blotting. The input contained 1% of the lysate used for immunoprecipitation. Asterisks (*) indicate non-specific signals from the antibodies (9D5 or NC). The amino acid numbers for human P0 are shown above.

Article Snippet: The isotype control antibody for 9D5 (mouse IgG2a κ) was purchased from Proteintech (65208-1-IG).

Techniques: Immunoprecipitation, Sequencing, Control, Western Blot