Mouse monoclonal ab against β actin

After washing with cold phosphate buffered saline
(PBS, Sigma-Aldrich, St. Louis, MO, USA),
the cells were lysed by a lysis buffer that contained
0.01 M Tris, pH=7.5, 0.1 M NaCl, 1% Triton
X-100, 0.5% sodium deoxycholate, and 0.1% sodium
dodecyl sulfate (SDS), with added protease
inhibitors (all purchased from Sigma-Aldrich, St.
Louis, MO, USA). Total proteins in cell lysates
were separated by 10% SDS-polyacrylamide gel
electrophoresis (PAGE, Sigma-Aldrich, St. Louis,
MO, USA) and transferred to a polyvinylidene
fluoride blotting membrane (PVDF, Sigma-Aldrich,
St. Louis, MO, USA). The membranes were
blocked in blocking bovine serum albumin solution
(BSA, Sigma-Aldrich, St. Louis, MO, USA)
and incubated with mouse anti-VEGF monoclonal
antibody (1:200), mouse anti-BCL-2 monoclonal
antibody (1:500) and mouse anti-SURVIVINmonoclonal antibody (1:500) (all purchased from
Abcam, Cambridge, England, UK) for one hour at
room temperature. After washing, the membranes
were incubated for 45 minutes with horseradish
peroxidase (HRP) -linked goat anti-mouse IgG
(1:5000, Sigma-Aldrich, St. Louis, MO, USA).
The protein bands were visualized by enhanced
chemiluminescence (Sigma-Aldrich, St. Louis,
MO, USA). Mouse monoclonal Ab against β-actin
(Abcam, Cambridge, England, UK) was used as a
housekeeping gene control. Band intensities were
semi-quantitatively analyzed by Image J densitometer
(NIH, Bethesda, MD, USA).

After washing with cold PBS, the cells were lysed by a lysis buffer containing 0.01M Tris, pH 7.5, 0.1M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS), with added protease inhibitors. Total proteins in cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene fluoride (PVDF) blotting membrane (Sigma-Aldrich, St. Louis, MO, USA). The membranes were blocked in blocking solution BSA (Sigma-Aldrich, St. Louis, MO, USA) and incubated with mouse anti-Eg5/KSP monoclonal antibody (1:200), mouse anti-VEGF monoclonal antibody (1:200), mouse anti-Cyclin D1 monoclonal antibody (1:500), mouse anti-Bcl-2 monoclonal antibody (1:500), mouse anti-Survivin monoclonal antibody (1:500), mouse anti-ANG2 monoclonal antibody (1:200) (All were bought from Abcam, Cambridge, ENG, UK) for 1 hour at room temperature. After washing, the membranes were incubated for 45 minutes with horseradish peroxidase (HRP)-linked goat anti-mouse IgG (1:5000, Sigma-Aldrich, St. Louis, MO, USA). The protein bands were visualized by enhanced chemiluminescence (Sigma-Aldrich, St. Louis, MO, USA). Mouse monoclonal Ab against β-actin (Abcam, Cambridge, ENG, UK) was used as a housekeeping gene control. Band intensities were semi-quantitatively analyzed by Image J densitometer (NIH, Bethesda, MD, USA).