Bovine serum albumin (bsa)

To measure receptor expression levels for AT1R-APEX and β2AR-APEX during proximity-labeling experiments, stable cell lines used for the proximity-labeling experiment were seeded on 10 cm plates, cultured and doxycycline-induced in the conditions used for proximity-labeling experiments. Cells were washed in PBS and subsequently harvested by incubating with PBS with 0.05% EDTA, pH 7.4, for 10 minutes at 4 oC. Cells were collected and centrifuged at 1000 × RPM for 5 minutes and re-suspended in assay buffer (20 mM HEPES, 100 mM NaCl, pH 7.4).
AT1R-APEX expression was assayed using radioligand binding using 50 nM [³H]-olmesartan. Binding reactions were conducted in assay buffer with 1 mg/mL bovine serum albumin (Rockland), pH 7.4. Specific binding was determined by subtracting nonspecific binding (10 µM candesartan) from total binding (assay buffer). Reactions were incubated for 90 minutes at room temperature and subsequently washed (50 mM Tris, pH 7.4) and captured on GF-B glass fiber filters using a Brandel harvester (Brandel). β2AR-APEX expression level was assayed using [³H]-dihydroalprenolol at 10 nM and 10 µM propranolol was used to measure non-specific binding by same procedure. Protein concentrations were measured using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).

FLAG-tagged human UPF1 was purified from baculovirus as described⁵⁷ . Human FMRP was purified from BL21-CodonPlus (DE3)-RIPL E. coli using a GSTrap HP Column (GE Healthcare Life Sciences) on which the GST tag was removed and FMRP was released using PreScission Protease (GE Healthcare Life Sciences). Protein purity and concentration were determined after electrophoresis in SDS–polyacrylamide and Coomassie blue staining, where serial dilutions of bovine serum albumin (Rockland) provided concentration standards. Pull-downs were performed essentially as described⁵⁷ but using FLAG-tagged bacterial alkaline phosphatase (Sigma–Aldrich) as a negative control and anti-FLAG agarose beads (Sigma–Aldrich).