Anti p21cip

Cells (5 × 10⁵) were lysed in RIPA buffer (0.1% sodium dodecyl sulfate, 1% NP-40 and 0.5% sodium deoxycholate in PBS at pH 7.4) supplemented with a protease and phosphatase inhibitor cocktail. The proteins (100 μg) were loaded onto SDS-PAGE gels and electrophoretically transferred to nitrocellulose membranes (Amersham Bioscience). After blocking with 5% non-fat dry milk in TRIS buffer saline-Tween 20, the blots were incubated overnight at 4 °C with either 1:1000 anti-lamin A/C (Cell Signaling Antibodies) or 1:1000 anti-p21^Cip (Abcam) or 1:1000 anti-p53 (Abcam) primary antibodies, and then with appropriate horseradish peroxidase-conjugated secondary antibody. Proteins were detected using Enhanced Chemiluminescence reagents (ECL, Promega) and chemiluminescence was detected with a Chemidoc XRS detection system equipped with Image Lab Software for image acquisition (Bio-Rad). The quantification of protein bands was performed by determining the relative optical density using ImageJ software (National Institutes of Health, Bethesda, MD).

The reagents were procured as follows: MiniMACS™ Starting Kit (Miltenyi Biotech, Auburn, CA); miRNeasy mini kit, miScript Reverse transcription kit, miScript SYBR Green kit, Universal primer for amplifying miR-2909, Pfu polymerase (Fermentas, Vilnius, Lithuania); Qiaquick PCR purification kit (Qiagen, Valencia, CA); pBlue TOPO reporter vector, Lipofectamine 2000 and β Gal Assay Kit (Invitrogen,Carlsbad, USA); anti-KLF4 (Abcam), anti-p21^CIP, anti-SP1, anti-MYC, anti-CCND1, anti-β actin; Annexin V-FITC apoptosis kit (Sigma-Aldrich, St.Louis, MO, USA); pMIR-GFP Reporter Vector (Cell Biolabs, San Diego, CA, USA); miRCURY LNA™ miR-2909 power inhibitor (EXIQON, Denmark); PMIRH-2909 lentiviral construct (System Biosciences, CA, USA).