To study whether USP6NL was associated with β-catenin, 100 μg of total protein in lysis supernatant of HCT116 cells transfected with siUSP6NL was added into protein G-Agarose beads (Roche), and then immune-precipitated with anti-USP6NL (Abcam), anti-β-catenin (Abcam) or control IgG antibody overnight at 4 °C. USP6NL and β-catenin in the immune complex were immunoblotted using anti-USP6NL (NBPI-47264), anti-β-catenin (ab6301, Abcam), respectively according the western blot method. Same amount of protein in each group was reserved for input control.
To study the effect of USP6NL on β-catenin ubiquitination, after fully lysis of HCT116 cells transfected with siUSP6NL, 100 μg of total protein in supernatant was added into Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, sc-2003), and then immune-precipitated with 1 μg of IgG (Santa Cruz Biotechnology, sc-2027) or anti-β-catenin antibody (ab227499) overnight at 4 °C. Ubiquitination levels of β-catenin in precipitated immune-complexes were separated and quantified using Anti-ubiquitin antibody (ab7780) according the western blot.
Sun K., He S.B., Yao Y.Z., Qu J.G., Xie R., Ma Y.Q., Zong M.H, & Chen J.X. (2019). Tre2 (USP6NL) promotes colorectal cancer cell proliferation via Wnt/β-catenin pathway. Cancer Cell International, 19, 102.
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