RPS15 mutant proteins are incorporated into ribosomes. (A) Distribution of RPS15 proteins (wild-type and P131S and S138F mutants) fused to GFP in HEK293T cells. rRNA was stained with the RNA-binding dye pyronin Y (PY). Colocalization is shown in merge panels. Scale bars, 20 µm. (B) Immunofluorescence analysis of endogenous RPS15 in GFP-expressing HEK293T cells. Scale bars, 20 µm. (C) Isolation of ribosomes from HEK293T cells stably expressing the GFP-RPS15 constructs and subsequent western blot analysis using antibodies against EF2, GFP, RPS15, RPS6, RPL11, and GAPDH. Note that RPL11, EF2, and GFP detection was carried out with the same samples run in parallel on an identical blot. A representative western blot of 2 independent experiments is shown. (D) Total protein lysates from HEK293T cells stably expressing GFP or the GFP-RPS15 constructs were immunoprecipitated with anti-GFP antibody, and GFP, RPS6 (as a representative ribosomal protein of the 40S subunit), and RPL11 (as a representative ribosomal protein of the 60S subunit) were detected by immunoblotting. Note that RPL11 and RPS6 detection was carried out with the same samples run in parallel on an identical blot. A representative western blot of 2 independent experiments is shown.