Gtx108181

Protein expression was detected by immunoblot analysis. Cell lysates with equal protein content were prepared in SDS sample buffer, separated on NativePAGE Novexw Bis–Tris 4–16% gel for BN-PAGE analysis (Invitrogen; Thermo Fisher Scientific, Inc.) followed by transferring to polyvinylidene fluoride membranes. Proteins on the membrane were detected with specific primary antibodies and HRP-conjugated secondary antibodies. The following antibodies were used: P62 (1:2000; ab56416, Abcam, Cambridge, MA, USA), β-actin (1:1000; ab3280, Abcam, Cambridge, MA, USA), LC3II (1:1000; AP1802a, Abgent, San Diego, CA, USA), Fzd 1 (1:1000; GTX108181, GeneTex, Irvine, CA, USA), GAPDH (1:10000; GTX100283, GeneTex, Irvine, CA, USA), GSK3β (1:1000; BD610202, BD Biosciences, San Jose, CA), GSK3β Ser9 (1:1000; #9323s, Cell Signaling Technology, Inc.), β-catenin (1:1000; #9562s, Cell Signaling Technology, Inc.), β-catenin Ser33/37/41 (1:1000; #9561s, Cell Signaling Technology, Inc.), CD133 (1:1000; #5860s, Cell Signaling Technology, Inc.), CD44 (1:1000; ab157107, Abcam, Cambridge, MA, USA), Nestin (1:1000; ABD69, Millipore Sigma, Burlington, MA), SOX-2 (1:1000; ab97959, Abcam, Cambridge, MA, USA), and β-actin (1:1000; sc47778, Santa Cruz Biotechnology, Santa Cruz, CA). The signal of each target protein was visualized by incubation with ECL Reagent and exposure to X-ray film.