Anti β tubulin 10094 1 ap

Cells were lysed in cell lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 50 mM NaF, and 0.1% NP-40) supplemented with protease and phosphatase inhibitors. Total protein extracts of tissues were generated in radioimmunoprecipitation (RIPA) assay lysis buffer containing complete protease and phosphatase inhibitor cocktail. Protein content was quantified using the Pierce BCA Protein Assay Kit (#23225, Thermo Fisher Scientific). The proteins were separated by SDS-PAGE, electrophoretically transferred to polyvinylidene difluoride (PVDF) blotting membranes (#LC2002, Thermo Fisher Scientific), and subjected to routine immunoblot analysis with primary antibodies as follows: anti-Bcl-2 (#15071), anti-Mcl-1 (#94296), anti-VHL (#68547), anti-CRBN (#71810), and anti-Sufu (#2522) were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA); anti-Bcl-x_L (#ab32370), anti-Gli1 (#ab134906), and anti-Smoothened (#ab236465) were obtained from Abcam (Cambridge, CB2 0AX, UK); anti-GAPDH (#60004-1-Ig) and anti-β-tubulin (#10094-1-AP) were purchased from Proteintech Group, Inc. (Rosemont, IL 60018, USA). The results were quantified by densitometry using ImageJ software (NIH).

WB analysis (20 μg of liver protein) was performed using anti-AMPKα (#2532, Cell Signaling Technology, Danvers, MA, USA), P 53 (A0263, ABclonal, Wuhan, China), caspase 3 (13847, Abcam, Cambridge, MA, USA) and anti-β-tubulin (10094-1-AP, Proteintech, Rosemont, IL, USA) antibodies. These antibodies have all been shown to successfully cross-react with M. amblycephala proteins. The signals of WB were quantitatively assayed by ImageJ 1.44 image analysis software.
Total RNA was extracted from the liver using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and its RNA concentration was detected by a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Then, cDNA was synthesized using PrimeScript Ist strand cDNA synthesis kit (Takara, Tokyo, Japan). Finally, the expression levels of target genes were detected by real-time PCR with specific primers (Table 2) under the SYBGREEN-based Light Cycle 96 system (Roche, LC96). EF1α (elongation factor 1 alpha) gene was used as an endogenous control because of the non-significant changes in the Ct value between the treatments. The expression levels of target genes were normalized by the expression of EF1α, and the relative expression levels were calculated by 2^−ΔΔCt method [28 (link)].