Ab49878

Cell lysates were prepared with high salt lysis buffer (150 mM Tris-HCl [pH 7.9], 500 mM NaCl, 1% (w/v) Nonidet P-40). Immunoblotting was performed according to a standard procedure using the following primary antibodies: anti-GST (sc-138, Santa Cruz Biotechnology), anti-β-actin (ab6276, Abcam), anti-Aiolos (sc-101982, Santa Cruz Biotechnology), anti-GSPT1 (ab49878, Abcam), anti-DDB1 (ab97522, Abcam), anti-CUL4A (ab72548, Abcam), anti-ROC1 (ab86862, Abcam), and anti-CRBN (CRBN65 mAb, Celgene)^{34 (link)}.

Protein levels were measured using western blot. Animal tissues were homogenized in RIPA buffer (Thermo Fisher Scientific) containing Halt Protease Inhibitor Cocktail (Life Technologies). Protein concentrations were determined using the BioRad DC protein assay, and protein was loaded at 40 μg onto a 4%–15% Criterion™ TGX™ Precast Midi Protein Gel (BioRad). Western blot membranes were probed with primary anti-eRF1 antibody (ab31799; Abcam) or anti-eRF3a antibody (ab49878; Abcam). An antibody against β-actin (A5316; Sigma) was used as a loading control. Membranes were then incubated with IRDye secondary antibodies (Li-COR) and scanned using an Odyssey infrared system (Li-COR). Images were quantified using Image Studio (Li-COR).
hFIX protein level in mouse plasma was measured by enzyme-linked immunosorbent assay (ELISA) using Human Factor IX (FIX) ELISA Kit (ab188393; Abcam) following manufacture instructions.

Cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.4% Nonidet P-40 in PBS, and blocked with Blocking One (Nacalai Tesque). Blocking One was also used for dilution of antibodies. The following primary antibodies were used: anti-HA (901501, BioLegend), anti-Aiolos (sc-101982, Santa Cruz Biotechnology), anti-IRF4 (sc-6059, Santa Cruz Biotechnology), and anti-GSPT1 (ab49878, Abcam).