Hrp conjugated anti mouse and anti rabbit igg

After incubation in normoxia or for 6, 12, or 24 h under hypoxia, cells were lysed and the protein concentration was determined (10). Proteins were separated on 10% polyacrylamide gels and transferred onto a nitrocellulose membrane. The monoclonal antibodies used for blotting were: anti-N-cadherin (mouse, 1:3000), anti-E-cadherin (mouse, 1:10,000) and anti-GAPDH (mouse, 1:100,000) (all from BD Transduction, San-Jose, CA, USA), anti-vimentin (mouse, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Snail (rabbit, 1:500; Novus Biologicals, Little Town, CO, USA), anti-Twist (mouse, 1:50; Abcam, Cambridge, UK) and HRP-conjugated anti-rabbit and anti-mouse IgG (both 1:7000; Santa Cruz Biotechnology). Western blot signals were measured by densitometric scanning with an Azure C300 Intelligent Dark Box (Biosystems Inc., Dublin, CA, USA). Each Western blot was performed three times.

Patient-derived tissue biopsies and harvested cells were lysed in RIPA buffer (Beyotime) for 30 min on ice. The lysate was centrifuged at 20,000 ×g for 20 min at 4 °C. The supernatant was transferred, and protein concentration was determined using the BCA Protein Assay kit (Thermo Fisher Scientific). Equal amounts of total and nuclear proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and electro-transferred onto a nitrocellulose membrane. Membranes were incubated with antibodies to NHE1 (Santa Cruz Biotechnology), CyclinD1 (Abcam, Cambridge, UK), PCNA (Abcam), MMP-2 (Santa-Cruz), MMP-9 (Santa Cruz Biotechnology), ERK1/2 (Epitomics, Burlingame, CA), phospho-ERK1/2 (Epitomics). The secondary antibodies used for detection were HRP-conjugated anti-rabbit and anti-mouse IgG (Santa Cruz Biotechnology). Immunoreactive bands were detected by an enhanced chemiluminescence kit (EMD Millipore, Darmstadt, Germany) and results were quantitated using an image analyzer Quantity One System (Bio-Rad Laboratories, Hercules, CA). β-actin and LaminB (Santa Cruz Biotechnology) were used as loading control for total and nuclear fractions, respectively.