Uvi 1d software

CD8⁺ T cells were lysed with RIPA buffer (1 mM phenylmethylsulphonylfluoride [PMSF], 5 mM EDTA, 1 mM sodium orthovanadate, 150 mM sodium chloride, 8 μg/ml leupeptin, 1.5 % Nonidet P-40, 20 mM tris–HCl, pH 7.4) and 40 μg of each lysate were separated by SDS-PAGE and electroblotted onto a nitrocellulose filter. The filter was blocked overnight at RT with 10 % BSA in PBS containing 0.1 % Tween-20 (TBS) and incubated with anti-human JAK3 and anti-human MCL-1 Abs in 5 % BSA-TBS (clones B-12 and B-6 respectively; 1:250 dilution, Santa Cruz Biotechnology) overnight, at 4 °C. The membranes were washed in TBS-T 0.1 % and incubated for 1 h at RT with horseradish-peroxidase-conjugated sheep anti-mouse IgG at 1:5000 dilution in TBS-T 0.1 %. The membrane was then visualized using the ECL enhanced chemiluminescence system (Amersham, Little Chalfont, UK). The same membranes were then immunoblotted again with anti-actin antibody (clone MM2/193; 1:2000 dilution; Santa Cruz Biotechnology). Signal detection and densitometric analysis of the bands was performed using a UVIchemi (UVItec, Cambridge) digital imaging system and UVI1D software (UVItec, Cambridge).

Mouse cortical purified synaptosomes were lysed in modified RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1mM sodium orthovanadate and protease inhibitors) and quantified for protein content with BCA assay. Samples were boiled for 5 min at 95°C in SDS-PAGE sample buffer. Proteins were separated on 10% precast polyacrylamide gel (Bio-Rad) by means of SDS–polyacrylamide gel electrophoresis and then blotted onto PVDF membrane. Membranes were blocked for 1 h at room temperature with Tris-buffered saline-Tween (t-TBS: 20 mM Tris, pH 7.4, 150 mM NaCl, and 0.05% Tween 20) containing 5% (w/v) non-fat dried milk, and then probed with the following primary antibodies overnight at 4°C: rabbit polyclonal anti-GluA1 (1:1000), rabbit monoclonal anti-GluA2 (1:2000), mouse monoclonal anti-GluA3 (1:500), rabbit monoclonal anti-GluA4 (1:500). After three 5-min washes in t-TBS, membranes were incubated for 1 h at room temperature with the appropriate horseradish peroxidase-linked secondary antibodies. After three 10-min washes in t-TBS protein bands were detected with an ECL (enhanced chemiluminescence) western blotting detection system. Images were acquired using the Alliance LD6 images capture system (Uvitec, Cambridge, UK) and analyzed with UVI-1D software (Uvitec, Cambridge, UK).

The cells and skin tissues were extracted using RIPA buffer. Next, the cells and skin lysates were homogenized to yield equivalent amounts of proteins based on the protein concentration measurements performed with Bradford reagent (Bio-Rad, Hercules, CA, USA). The homogenized proteins were electrophoresed on 10–15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred from SDS-PAGE to a nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). Non-specific binding was blocked with 5% bovine serum albumin in TBST (50 mmol/1 Tris-HCL, pH 7.5, 150 mmol/1 NaCl, and 0.1% Tween 20) for 1 h at room temperature followed by overnight incubation of membranes with primary antibodies at 4 °C. The membranes were washed with TBST thrice and incubated with a secondary antibody (Santa Cruz Biotechnology Inc., TX, USA) for 1 h at room temperature. Finally, the proteins were visualized using a chemiluminescence detection ECL reagent (GE Healthcare Life science, MO, USA) and quantified with UVI-1D software (UVITEC, Warwickshire, UK).

Samples were prepared as previously described [28 (link)], resolved on Bolt pre-cast 4-12% gels (Thermo Fisher Scientific), using Bolt MES SDS running buffer (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes. After a blocking step in 5% non fat-dried milk in phosphate-buffered saline (PBS)-0.1% Tween, membranes were incubated with primary antibodies overnight at 4 °C. After three washes in PBS - 0.1% Tween, membranes were incubated with the appropriate HRP-linked secondary antibodies (Bio-Rad Laboratories) for 45 min at room temperature, washed with PBS-0.1% Tween and analysed by chemi-luminescence (GE Healthcare Life Science). Images were acquired and quantified using Alliance Mini HD6 system by UVITEC Ltd., Cambridge, equipped with UVI1D Software (UVITEC, 14-630,275). Detailed information for all antibodies is provided in the Supplementary Table S1.

Cells were treated as described in Bruno T. et al., 2006 (44 (link)). Samples were separated by electrophoresis and transferred onto nitrocellulose membranes. After a blocking step in 5% non-fat-dried milk in 0.1% Tween-PBS, membranes were incubated with primary antibodies overnight at 4°C. After three washes in 0.1% Tween-PBS, membranes were incubated with the appropriate HRP-linked secondary antibodies (Bio-Rad, IT) at room temperature for 45 min, washed with 0.1% Tween-PBS and analyzed by chemi-luminescence (GE Healthcare Life Science, IT). Images were acquired using Alliance Mini HD6 system by UVITEC Ltd, Cambridge, equipped with UVI1D Software (UVITEC, 14–630275). The primary antibodies used were: anti-Che-1 (43 (link)), and anti-β-actin (Sigma – Aldrich, Merck, IT).