Ab139699

Antibodies against alpha-tubulin (ab15246) and KLF2 (ab139699) were obtained from Abcam (Cambridge, MA). Antibodies against acetylated-alpha-tubulin (sc23950) were obtained from Santa Cruz (Dallas, TX). Actin (A2066) antibodies were obtained from Sigma-Aldrich (St. Louis, MO). Procedures were performed as previously described^{16 (link)}.

Cell lysate was subjected to 12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. Rabbit polyclonal antibody against KLF2 (ab139699, Abcam, Cambridge, MA, USA) and p21 (C-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as primary antibodies. Secondary antibody was purchased from Cell Signaling (Beverly, MA, USA). Protein bands were visualized using an enhanced chemiluminescence detection system.

HAECs were lysed in RIPA buffer (Sigma-Aldrich) including protease inhibitors (Complete Protease Inhibitor Cocktail, Roche). Cell lysates were resolved on SDS–PAGE gels and then transferred to nitrocellulose membranes. Proteins were detected using primary antibodies against KLF4 (1:1,000, EPR3550(2)ABC, Abcam), KLF2 (1:75, ab139699, Abcam) and GAPDH (1:1,000, clone 6C5, Millipore), and horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,000, Goat Anti-Mouse IgG HRP Affinity Purified PAb, R&D Systems; Goat Anti-Rabbit IgG HRP Affinity Purified PAb, Santa Cruz). Protein bands were visualized using an enhanced chemiluminescence detection system (ECL Advance, GE Healthcare Life Sciences) and an LAS 3000 Imager (Fuji Photo Film Co., Ltd.) and were quantified using Multigauge software (Fuji Photo Film). Intensities of the KLF4 and KLF2 bands were expressed as a percentage of those of the GAPDH bands.