Anti cyclin d1

The cells or tissues were treated with RIPA lysate, properly mixed, centrifuged, and the supernatant was added to electrophoresis loading buffer. The separated proteins in the gel were transferred to PVDF membranes, coated with primary antibody overnight, incubated with secondary antibody the next day, and then exposed and developed using a gel imaging system. Primary antibodies used were anti-VDAC1 (A19707; Abclonal), anti-β-Catenin (A0316; Abclonal), phosphorylated β-catenin (AP1076; Abclonal), anti-Cyclin D1 (A0310; Abclonal), anti-GAPDH (A19056; Abclonal).

Cells were cultured at a density of 1.5 × 10⁵ cells/well on 8 mm coverslips in 12-well plates. After 48 hours, coverslips were fixed by ice-cold methanol, and incubated with primary E-cadherin (Abcam), Vimentin and β-catenin (Epitomics) antibodies prior to florescent-labeled secondary antibodies. Nuclear DNA was stained with 4′, 6-diamidino-2-phenylindole (DAPI) and coverslips were mounted with FluorSave reagent (CALBIOCHEM). Immunofluorescence images were taken by Olympus inverted fluorescence microscope and were outputted by PV10-ASW 1.7 viewer software. Immunoblotting was performed as follows: Proteins were extracted with lysis buffer and then quantified by the BCA method (KeyGen Biotech). Lysates were diluted in SDS sample buffer (KeyGen Biotech) prior to SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Roche Applied Sciences). Membranes were immunoblotted overnight at 4°C with anti-SOX17 (Millipore), anti-CyclinD1 (Abclonal), anti-C-myc, anti-DKK1 (Cell Signaling Technology), anti-E-cadherin (Abcam), anti-SOX2, anti-β-catenin, anti-Vimentin and anti-Slug and anti-N-cadherin antibodies (Epitomics), followed by the appropriate second antibodies. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Gel densitometry (Bio-Rad) was used to quantify immunoblot signals on exposed film.

Cells were lysed by iced radioimmunoprecipitation assay (RIPA) buffer (50mM Tris pH 7.4, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1mM ethylenediamine tetraacetic acid (EDTA), 0.1% SDS) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentration was measured by bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China). Protein samples were subjected to SDS-PAGE electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% skim milk in Tris-buffered saline plus Tween 20 (TBST) for 1h, the membranes were incubated with primary antibodies at 4 ℃ overnight, and then incubated with corresponding radish peroxidase-conjugated secondary antibodies (Proteintech, Wuhan, China) for 1 h at room temperature. Western blot bands were detected via West Pico Super Signal chemiluminescent substrate (Pierce, Rockford, IL, USA) using ChemiDoc XRS Imaging System (BioRad, Hercules, CA, USA). SRSF1 and β-actin antibodies were purchased from Santa Cruz. Variants were detected with anti-VEGF (Abcam, No.ab1316), anti-CD44 (ABclonal, No. A1351), and anti-Cyclin D1(CST, No. 2926P) antibodies, respectively. For relative quantification, the integrated optical density (IOD) was estimated using ImageJ (NIH). Relative protein expression level was calculated as IOD Experimental/IOD Control.