Anti flag dykddddk tag

An anti-IGF2BP2 antibody (1–2 mg per test, Abcam, ab124930) and an anti-FLAG/DYKDDDDK Tag (1–2 mg per test, Cell Signaling Technology, 8146 s) were used in the IP assays, and the proteins were detected by Western blotting with an anti-ubiquitin antibody (1:1000, Cell Signaling Technology, #3933) according to the manufacturer’s instructions.

In vitro ubiquitination assays were performed in a 40 µl volume with 0.15 μM UBA1, 1 μM CDC34, 1 μM neddylated CUL2-RBX1 or 1 μM CUL2-RBX1, 1 μM FEM1B-ELOB-ELOC, 1 μM Flag-CCDC89 and 30 μM Ub at 37 °C in reaction buffer (25 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 1 mM DTT and 5 mM ATP). The reactions were terminated by adding 5× sodium dodecyl sulfate (SDS) sample buffer and then resolved by SDS–PAGE. Ubiquitinated products were detected by immunoblotting. The same concentrations of FEM1B and Flag-CCDC89 variants were used in the assay as the respective wild type. The following antibodies were used: anti-FLAG DYKDDDDK tag (Cell Signaling Technology, Cat#2368, 1:1,000 dilution) and HRP-linked anti-rabbit IgG (Cell Signaling Technology, Cat#7074, 1:1,000 dilution). The experiments were performed three times with similar results, and the quantification was performed using ImageJ, with the data subsequently analyzed with  GraphPad Prism 8.

IP assays were conducted with anti-FLAG/DYKDDDDK Tag (Cell Signaling Technology, USA, 1–2 mg each test) together with anti-PSPC1 antibody (Abcam, USA, 1–2 mg each test), and anti-ubiquitin antibody (American Cell Signaling Technology, 1:1000) was utilized to identify the protein through Western blotting in accordance with the instructions of manufacturer.