Anti α tubulin

Indirect immunofluorescence was performed as previously described [5 (link)] by using the anti-EB1 antibody and anti-mouse antibody Alexa 568 nm (Molecular Probes); and FITC-coupled anti-α-tubulin antibody (clone DM1A; Sigma-Aldrich). Cells were observed using a Leica DM-IRBE microscope, 100X magnification. All images were acquired using Metamoph software (Molecular Devices, Sunnyvale, CA) at identical acquisition settings, and were processed using Image J software. After cell lysis, 30 μg of total protein were loaded into a 12% SDS-PAGE gel. Anti-EB1 antibody, anti-α-tubulin and anti-mouse IgG-horseradish peroxidase (Jackson Immunoresearch) were used. Chemiluminescence detection kit (Millipore) was used for visualization of protein bands. Chemiluminescent signal was acquired on a G:BOX imaging system (Syngene, Cambridge, UK) and quantification was done with Image J software.

Protein lysates were prepared in RIPA lysis buffer with protease inhibitor cocktail (Roche), lysates were separated by SDS-PAGE, and the proteins were transferred to a PVDF membrane (Millipore). The membrane was reacted with the following primary antibodies at 1:1000: anti-p53 antibody (DO1) (Santa Cruz, sc-126), anti-eNOS (BD Transduction, 610296), anti-phospho-eNOS (ser1177) (Cell Signaling Technology, CST9571), anti-β-actin (Cell Signaling Technology, 4970), and anti-α-tubulin (Cell Signaling Technology, 2125). Blocking was done with 5% skim milk. The secondary antibodies were horseradish peroxidase-conjugated anti-mouse immunoglobulin-G (Jackson, 115-035-003) for anti-p53 and anti-eNOS, as well as horseradish peroxidase-conjugated anti-rabbit immunoglobulin-G (Jackson, 115-035-144) for anti-phospho-eNOS, anti-β-actin and anti-α-tubulin. Secondary antibodies were added at a concentration of 1:5000. Proteins were detected by chemiluminescence using ECL western blotting detection reagents (GE Healthcare, UK). Relative protein levels were quantified with Image J software and were normalized by using β-actin or α-tubulin as loading controls.