α cyclin d1

Cells were lysed using lysis buffer (40 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% sodium dodecyl sulfate (SDS)). Approximately 20 μg of the protein extract was resolved via SDS-PAGE and analyzed via immunoblotting with primary antibodies against α-HIF-1α (Cell Signaling, Danvers, MA, USA, 3716), α-PARP (Cell Signaling, 9542), α-Caspase-3 (Cell Signaling, 9662), α-p44/42 MAPK (ERK1/2) (Cell Signaling, 4695), α-Phospho-p44/42 MAPK (Erk1/2) (Cell Signaling, 4370), α-p38 MAPK (Cell Signaling, 9212), α-Phospho-p38 MAPK (Cell Signaling, 4511), α-JNK2 (Cell Signaling, 9258), α-Phospho-SAPK/JNK (Cell Signaling, 4668), α-Cyclin B1 (Santa Cruz, Dallas, TX, USA, sc-752), α-Cyclin D1 (Cell Signaling, 2978), α-Cyclin E1 (Cell Signaling, 4129), α-CDK4 (Cell Signaling, 12790), α-p53 (Millipore, Burlington, MA, USA, 05-224), α-p21 (Cell Signaling, 2947), α-TBP (Abcam, Cambridge, UK, ab818), α-β-actin (Santa Cruz, sc-47778), and α-alpha-tubulin (Abcam, ab7291).

The whole cell proteins of MDA-MB-231/MDA-MB-468 cells were extracted from cells using RIPA buffer (Beyotime, Shanghai, China) and the nuclear extracts were prepared with a NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology, Rockford, USA). Western blots were probed with α-phospho LRP6 (Ser1490), α-LRP6, α-β-catenin, α-E-cadherin, α-N-cadherin, α-Vimentin, α-Snail, α-Histone H3, α-c-Myc, α-cyclin D1, α-Axin, α-CD44, α-HIF-1a, α-Glut1 (Cell Signaling Technology, USA), α-VEGFA (Abcam, Cambridge, UK), α-β-actin.