Extended Data Fig. 9: isASO-ID. Dose-dependent changes in the ASO-protein interactome.

a)-f) SILAC-MS/MS for BG-ASO (20mer MOE) at increasing concentrations (15 nM, 25 nM, 50 nM, and 100 nM) against the control ASO. The amount of BG-ASO was constant for all transfections at 15 nM and conjugation-incompetent control ASO (20mer MOE) was spiked in to obtain the final ASO concentration to promote equal amounts of bound SNAP-BASU-His in all conditions. Plotted was the normalized enrichment (log2) of replicate 1 against replicate 2 with swapped SILAC labels. a) 15 nM BG-ASO over 25 nM control ASO. b) 15 nM BG-ASO and 10 nM control ASO over 25 nM control ASO. c) 15 nM BG-ASO and 10 nM control ASO over 15 nM BG-ASO. d) 15 nM BG-ASO and 35 nM control ASO over 100 nM control ASO. e) 15 nM BG-ASO and 85 nM control ASO over 100 nM control ASO. f) 15 nM BG-ASO and 85 nM control ASO over 15 nM BG-ASO and 35 nM control ASO. g) Four stacked plots. 15 nM BG-ASO over 25 nM control ASO (dark blue), 15 nM BG-ASO and 10 nM control ASO over 25 nM control ASO (light blue), 15 nM BG-ASO and 35 nM control ASO over 100 nM control ASO (orange), and 15 nM BG-ASO and 85 nM control ASO over 100 nM control ASO (red). Changes between the enrichments are indicated with arrows for panels g and h (blue for depletion upon increased ASO dose, red for further enrichment upon increased ASO dose). Highlighted are known ASO interactors and proteins with notable changes. For the isASO-ID SILAC-MS/MS experiments in panel a-f, detected proteins were colored according to their biological function as P-body component (gold), paraspeckle protein (red) and the annotated GO terms DNA binding (orange) and RNA binding (blue). Ribosomal proteins were colored in purple. Proteins in panels a-f passing the applied enrichment threshold (>1 log2 unit) are represented as filled squares. Carboxylases are highlighted as filled black squares and represent unregulated control proteins.