P45 99469

Cells (1 × 10⁷) were incubated with 2 μg FLAG (F1804; Sigma), AGO2 (SAB4200085; Sigma), anti‐AUF1 (P45‐99469; Thermo), or IgG primary Abs for 4 h at room temperature. Protein G was then added to each sample, then analyzed by qRT‐PCR and western blot.

To detect colocalization of circARHGAP5 with indicated proteins, cells were incubated with primary Ab (1:200 dilution, P45‐99469; Thermo Fisher Scientific) for 4 h at room temperature, which was followed by a reaction with Alexa Fluor 488‐conjugated secondary Abs (1:100 dilution; Life Technologies). Subsequently, hybridization using Cy‐3‐conjugated circARHGAP5 probes was carried out at 37°C in the dark overnight and the nuclei were stained with DAPI (Dojindo Laboratories). Visualization of the staining was carried out with a Zeiss LSM 880 confocal laser scanning microscope. Probe sequence is shown in Table S3.

For western blots, samples were separated on SDS‐PAGE gels. Membranes were incubated with Ab anti‐FLAG (1:1000 dilution, 20,543‐1‐AP; Proteintech), AUF1 (1:2000 dilution, P45‐99469; Thermo Fisher Scientific), AGO2 (1:1000, SAB4200085; Sigma), BIM (1:600 dilution, Proteintech, 22,037‐1‐AP), or anti‐β‐actin (1:1000 dilution, HC201; TransGen).