Optimization of RNase treatment of testis extracts before co-immunoprecipitation.

(A) Non-denaturing RNA agarose gel showing multiple RNase treatment conditions (lanes 3–6, see box for details) or untreated BL/6 testis total RNA (lane 2) as a control; the treatment used in lane 5 was chosen for co-immunoprecipitation experiments. Approximately 2 μg of RNA were loaded on each lane. (B) Bioanalyzer traces of RNA samples corresponding to lanes 2 and 5 in panel A. RIN: RNA Integrity Number; scale from 1 (fully degraded) to 10 (intact). (C) Silver staining of anti-ORF1p co-immunoprecipitation samples (IP) from Mael^-/- testicular extracts minus (-) and plus (+) the RNase treatment used for lane 5 in panel A. Immunoprecipitation with an isotype IgG served as a negative control; note that the RNase treatment increases immunoprecipitation of ORF1p. (D) Heatmap showing the recovery of ribosomal proteins in anti-ORF1p co-immunoprecipitation samples from Mael^-/- testes, minus (-) and plus (+) the RNase treatment used for lane 5 in panel A. Color indicates protein levels; E2.n indicates Experiment 2.replicate n. The relative abundance of each protein in each eluate was obtained by mass spectrometry analysis and further adjusted by dividing the PSM counts by molecular weights (MW); for a sample-to-sample comparison, the obtained PSM / MW ratios were normalized to the ratio obtained for the bait (LORF1).

(TIF)