Procaspase 9

Cells were treated with CP and, after different times, were collected, centrifuged and washed with ice cold phosphate-buffered saline (PBS). The pellet was then resuspended in lysis buffer as described [47 ]. The protein concentration in the supernatant was determined using the BCA protein assay (Pierce, Italy). Equal amounts of protein (10 μg) were resolved using SDS-PAGE gel electrophoresis (Criterion precast Tris-HCl gel, BioRad, Italy) and transferred to PVDF Hybond-p membranes (GE Healthcare, Italy). Membranes were blocked with 2% ECL-Blocking Solution (GE Healthcare, Italy) for 2 hours at room temperature. Membranes were then incubated with primary antibodies against Bcl-2, PARP, procaspase-9, cleaved caspase-7, GRP78, (Cell Signaling, Italy), cleaved caspase-12 (Abcam, UK), β-actin (Sigma Aldrich, Italy), and cleaved caspase-3 (Novus Biologicals, Italy) overnight. Membranes were then incubated with peroxidase-conjugated secondary antibodies (Invitrogen, Italy) for 60 min. All membranes were visualized using ECL Select (GE Healthcare, Italy) and exposed to Hyperfilm MP (GE Healthcare, Italy). To ensure equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.

Rabbit anti-human Bax, ERK, JNK and p-JNK antibodies were obtained from ProteinTech Group (Chicago, IL, USA). Rabbit anti-human antibodies against p-PI3K, AKT, p-AKT, PARP, pro-caspase-3, cleaved-caspase-3, pro-caspase-9, cleaved-caspase-9, Bcl-xl, Bcl-2, Mcl-1, Bad, p38, p-p38, p-ERK, p-ATF2 and p53 were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-human antibodies against p-Bad and cytochrome C were obtained from Epitomics (Burlingame, CA, USA). Rabbit anti-human β-actin antibodies were obtained from Sigma (St Louis, MO, USA). Goat anti-rabbit and horseradish peroxidase conjugated IgG was obtained from Millipore (Bellerica, MA, USA). Immunoblotting was performed as previously described [19 (link)]. The treated samples and the control samples (25 μg) were separated by 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins on the gel were then transferred to PVDF membranes. The PVDF membranes were incubated with the primary antibody (1:1000 dilutions in 2% dehydrated skim milk) at 4 °C overnight. Then, incubation at 4 °C was performed for 2 h with the secondary antibodies (goat anti-rabbit or goat anti-mouse and horseradish peroxidase conjugate, 1:5000 dilution in 2% dehydrated skim milk). The blots were detected through chemiluminescence using enhanced ECL western blotting kit.

The cells were subsequently lysed in RIPA solution containing protease inhibitors (Roche). The total extracted protein content (25 μg) was separated by SDS-PAGE and transferred to polyvinyl difluoride (PVDF) membranes. The PVDF membranes were incubated by primary antibodies at a dilution of 1:500 or 1:1000 to detect procaspase-9, caspase-9, procaspase-3, caspase-3 (Cell Signalling), COX3, GAPDH (Santa Cruz), collagen I, collagen III, BAX, BAK, Bcl-2, Bcl-xL, Bcl-2-associated death promoter (BAD), p53 upregulated modulator of apoptosis (PUMA), β-actin, Apaf-1, Cyt c, EndoG, AIF (GeneTex) and α-SMA (abcam). The fold change in protein expression was expressed as a ratio calculated by dividing the specific protein band density by the β-actin band density.

A detailed protocol has been published previously [3 (link),11 (link)]. In brief, cells were incubated with different concentrations of AMF for the indicated time periods. Cells were then collected, washed with PBS and lysed. In all experiments, whole cell extracts were used. Fifty μg of protein per lane were then resolved using a gradient Bis-Tris gel (Invitrogen, Leek, The Netherlands) and transferred onto a nitrocellulose membrane (Invitrogen). After blocking for 1 hour using Starting Block (TBS) buffer (Pierce Biotechnology, Rockford, IL), primary antibody was added and incubated overnight at 4°C. Fluorescence-labeled secondary antibodies (Molecular Probes, Eugene, OR) were used. The membranes were scanned and quantified using the Odyssey infrared imaging system (LiCor Biosciences, Lincoln, NE). Antibodies were purchased from the following companies: beta-actin (Chemicon Int., Temecula, CA), cleaved PARP (Promega, Madison, WI), pro- and cleaved caspase-3 (Upstate Cell Signaling Sources, NY), pro- and cleaved caspase-8 (Naga-ku Nagoya, Japan), pro-caspase-9 (Cell Signaling Technology Inc, Beverly, MA).

Minimum essential medium (MEM) and fetal bovine serum (FBS) were purchased from Invitrogen (CA). Antibodies specific to Bcl-2, pro-caspase-3, pro-caspase-8, pro-caspase-9, cleaved-caspase-3, Bax, poly ADP-ribose polymerase (PARP), Bid, truncated Bid (t-Bid), androgen receptor (AR), prostate-specific antigen (PSA), phosphatase and tensin homolog (PTEN), anti-microtubule-associated protein light chain-3 (LC3), and beclin1 were purchased from Cell Signaling Technology Inc. (MA). 3-Methyladenine (3-MA) was obtained from Sigma Aldrich (MO). SLE was extracted with 100% ethanol at room temperature for 24 hours in a shaker, and the filtered extracts were concentrated and powdered under reduced pressure. The powder was lyophilized and stored at 4°C.