Ab65222

The cells were collected using cell lysissolution (Sigma) and denatured at 100 °C for 5 min. The protein concentrations were tested using bicinchoninic acid (BCA) Protein Assay Kit (Pierce, USA). Dodecyl sulphate polyacrylamide gel (SDS-PAGE) electrophoresis was performed to separate the proteins. Next, the proteins were transblotted onto nitrocellulose membranes (Amersham, USA). Primary antibodies were added after the membranes have been blocked with 5% not-fat milk. Then, the membranes were maintained at 4 °C overnight. Primary antibodies were as follows: anti-Col2A1 (1:8000, ab34712,abcam), anti-E2F2 (1:1000, ab65222), anti-p16INK4a (1:5000,ab108349) and anti-β-actin (ab8226,1:5000). Secondary antibodies (abcam) were incubated at room temperature for 2 h. The blot bands were developed with Enhanced chemiluminescence (Amersham).The density of bands was quantified using Quantity one 4.6.2.

Cells were seeded in 48 well plate with confluence of 50%. Then cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 10 min after indicated treatment. 5% BSA was used for blockage for 1 h at room temperature. Cells were then incubated with primary antibodies against E2F2 (1:200, ab65222, Abcam, USA) at 4 °C overnight. After being washed with PBS three times, cells were incubated with Alexa Fluor 488-conjugated secondary antibodies (1:500, Invitrogen) for 1 h at 37 °C. The cells were mounted with prolong gold antifade reagent with DAPI (Invitrogen) for 10 min. After rinsed with PBS for three times, samples were analyzed with confocal fluorescence microscope (Olympus, Japan).