Anti kif3a

The concentrations of protein samples extracted from the tissue specimens and cell lines in radioimmunoprecipitation assay lysis buffer were determined using the Bradford reagent (Sigma) according to the manufacturer’s instructions. Western blot analysis was performed using a standard protocol. The following antibodies were used: rabbit anti-Tg737 (1:2,000; Protein Tech), anti-Kif3a (1:2,000; Abcam), anti-SREBP2 (1:3,000; Abcam), anti-HMGCR (HPA008338; Sigma), antitotal ERK (1:1,000; Cell Signaling Technology), antiphosphorylated ERK (1:1,000; Cell Signaling Technology), anti–β-catenin (1:1,000; Cell Signaling Technology), anti-Ras (1:3,000; BD Bioscience), antitubulin (1:3,000; Santa Cruz), and anti-GAPDH (1:3,000; Santa Cruz). Total RNA was extracted from the cells using TRIzol according to the standard protocol. 2 µg RNA was processed directly into cDNA using a reverse transcription kit (Promega) according to the manufacturer’s instructions. Amplification reactions were performed and relative gene expression levels were calculated as previously described (Deng et al., 2007 (link)). The primers used in these experiments are listed in Table S5.

The procedure of IHC staining was performed as described previously. Anti–KIF3A (abcam, ab11259, dilution at 1:300), anti–phospho‐Rb, anti–E2F1, anti–Cyclin E1, anti–Cyclin D1, anti–P21, anti–ZEB1, anti–E‐cadherin, anti–vimentin, anti–MMP‐9 and anti–MMP‐2 (Bioworld Technology, all at dilution 1:200) were incubated overnight at 4°C. Each slide was photographed with a digital camera on an inverted Olympus IX81 microscope. The staining evaluation was made according to the semi–quantitative scoring system, as described previously. Briefly, the scores of positive tumor cell proportion (0, none; 1, <1/100; 2, 1/100 to 1/10; 3, 1/10 to 1/3; 4, 1/3 to 2/3; and 5,> 2/3) and staining intensity (0, none; 1, weak; 2, intermediate; and 3, strong) were added up to obtain a final total score, ranging from 0 to 8.