Dot blot vacuum apparatus

In mantle and PAM samples, a dot blot apparatus was employed for the determination of cleaved caspases and ubiquitin conjugate levels. Samples were diluted in a saline solution in a concentration of 5 μg mL⁻¹ (150 mM NaCl). Subsequently, equal sample volumes (100 μL) were loaded in a dot blot vacuum apparatus (BioRad) and gravity-fed through a pre-soaked nitrocellulose membrane (0.45 μm), which was thereafter blocked at room temperature for 30 min with 5% (w/v) non-fat milk in TBST (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% (v/v) Tween 20). The derived nitrocellulose membrane was then incubated with the following antibodies according to the manufacturer’s instructions: anti-cleaved caspase antibody (8698, Cell Signaling, Beverly, MA, USA) and anti-ubiquitin antibody (3936, Cell Signaling, Beverly, MA, USA). The dots were washed with TBST, incubated with horseradish peroxidase-linked secondary antibodies and washed again with TBST. Thereafter, enhanced chemiluminescence (Chemicon) with exposure to Fuji Medical X-ray films was employed in order for the dots to be detected. Laser-scanning densitometry (GelPro Analyzer Software, GraphPad, USA) was employed for films’ quantification.

Cleaved caspases and ubiquitin conjugate levels were determined in mantle and PAM samples with the employment of a dot blot apparatus. Specifically, samples were diluted to a concentration of 5 μg mL⁻¹ in a saline solution (150 mM NaCl); 100 μL volumes were loaded onto a pre-soaked nitrocellulose membrane (0.45 μm) in a dot blot vacuum apparatus (BioRad), and gravity-fed through the membrane. The membrane was blocked with 5% (w/v) non-fat milk in TBST (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% (v/v) Tween 20) for 30 min at room temperature. The resulting nitrocellulose membrane was subjected to overnight incubation with anti-cleaved caspase antibody (Cat. No.8698 Cell Signaling, UK) and polyclonal anti-ubiquitin rabbit antibody (Cat. No. 3936, Cell Signaling, UK). Antibodies were diluted as recommended by the manufacturer’s guidelines. After washing in TBST (3 periods, 5 min each time), the dots were incubated with horseradish peroxidase-linked secondary antibodies, washed again in TBST (3 periods, 5 min each time), and the dots were detected using enhanced chemiluminescence (Chemicon) with exposure to Fuji Medical X-ray films. Films were quantified by laser-scanning densitometry (GelPro Analyzer Software, GraphPad, USA).