Horseradish peroxidase linked secondary antibody

Western blotting was performed as described previously [25 (link)]. Briefly, whole-cell lysates from MC3T3-E1 cells were mixed with Laemmli’s buffer, separated by 5–20% SDS-polyacrylamide gel electrophoresis, and transferred onto PVDF membranes. The following primary antibodies were used: anti-PAR (Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (Sigma-Aldrich). Immune complexes were visualized using horseradish peroxidase-linked secondary antibodies (Cytiva, Tokyo, Japan). Protein bands were visualized with an enhanced chemiluminescence kit (Merck, Branchburg, NJ, USA). Image quantification was performed with Image J software (NIH, Rockville, MD, USA).

UN-ADC12, UN-ADC18, UN-SCC679, and UN-SCC680 cell lines were seeded at 300 000 cells per well in 6-well plates and allowed to attach overnight. Cells were treated with PBS, sunitinib (at concentrations ranging from 100 nM to 1 μM), DC101 (100 μg/ml), or rat IgG1 antibody (100 μg/ml) for 48 h. Cells were lysed and total proteins were extracted as previously described (Catena et al, 2010 (link)). Twenty μg of total protein from each lysate was boiled at 95°C for 5 min, separated by SDS–PAGE under reduced conditions (5% 2-mercaptoethanol), and transferred onto nitrocellulose membranes. The membranes were subsequently blocked in 5% defatted milk–PBS for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-AKT (1:1000; 9272; Cell Signaling Technology, Beverly, MA, USA), anti-pAKT (1:1000; 9271; Cell Signaling Technology), anti-ERK (1:1000; 9102; Cell Signaling Technology), and anti-pERK (1:1000; 9101; Cell Signaling Technology). Blots were then incubated with a horseradish peroxidase-linked secondary antibody (1:2000; Amersham Pharmacia Biotech, Little Chalfont, UK) and developed by chemiluminescence with Lumilight plus kit (Roche diagnostics, Burgess Hill, UK).

The homogenates were run on 4–20% SDS–acrylamide, and then electroblotted onto PVDF membrane (Hoefer Scientific Instruments, San Francisco, CA, USA) and immunostained with primary antibodies and horseradish peroxidase-linked secondary antibody (Amersham Biosciences, UK). The signal was detected by fluorescence using ECF substrate (Amersham Biosciences, UK). The fluorescence signals were visualized by a fluorescence digital camera detection system (Typhoon and Kodak scanner).