Casepase3

786-0 and Caki-2 cells were washed twice with PBS, and were collected in a lysis buffer. Membranes were blocked with 5% skim milk, followed by incubation with antibodies against ARHGAP24, Casepase3, PCNA, CDK1, CDK2 (Abcam, Cambrige, MA), Bax, Bcl-2(Santa Cruz, CA, USA) and GADPH (CST, Beverly, USA). After incubating with secondary antibodies, the signals were visualized using enhanced chemiluminescence (ECL, Millipore).

Cells were stimulated with LPS for 0.5, 1, and 1.5 h after being pretreated with CA for 3 h; then, the proteins were extracted using a total protein extraction kit (BioChain Institute Inc., Hayward, CA) and quantified using a BCA protein assay kit (Pierce Biotechnology Inc., IL). Western blot analysis was performed using equal quantities (10-20 μg) of cell extracts, diluted in sample buffer, and separated using 4-12% gradient SDS-PAGE gels. Then, proteins were transferred to PVDF membranes (Millipore, Biotechnology Inc.) and hybridized with specific antibodies. The following primary antibodies were from Cell Signaling Technology, Danvers, MA: c-Jun NH2-terminal kinase 1/2 activation (JNK, #9258), phospho-JNK (#4668), p38 (#8690), phospho-p38 (#4511), extracellular signal-regulated kinase (ERK, #4695), phospho-ERK (#4370), c-Jun (#9165), phospho-c-Jun (#3270), and β-actin (#4970). The following primary antibodies were from Abcam, Cambridge, UK: Bcl-2 (ab183656), Bax (ab32503), and casepase-3 (ab90437). The stripes were detected using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). In all cases, β-actin was used as a loading control. Densitometric values of immunoblot signals were quantified using ImageJ.