Nitrocellulose membranes 0.45 μm

Equal amounts of protein were separated by SDS-PAGE in 4–12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes (0.45 μm, GE healthcare, Little Chalfont, UK). The filters were blocked in TBS-T containing 5% non-fat dried milk for 1 h at room temperature or overnight at 4 °C. Proteins were visualized using appropriate primary antibodies. All primary antibodies were diluted in TBS-T and incubated with the nitrocellulose blot overnight at 4 °C. Incubation with secondary peroxidase coupled anti-mouse or anti-rabbit was performed by using the ECL system (Thermo Scientific SuperSignal West Pico, 34080; Amersham ECL Prime, RPN2232). The nitrocellulose membrane was then stripped with buffer Restore (Pierce, Rockford, IL) and subsequently re-probed with the specific antibodies for the unphosphorylated proteins. GRB2 was used as the loading control for all markers. The level of phosphorylation was calculated as the ratio between data from anti-phospho antibodies over those from the related not phosphorylated counterparts. Protein loading was monitored by normalization to β-actin and growth factor receptor-bound protein 2 (GRB2) [52 (link)]. Blots were scanned and quantitative densitometric analysis was performed by using ImageJ (http://imagej.nih.gov/ij/).

Cells were lysed in ice-cold RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM EDTA) containing a combination of protease and phosphatase inhibitor cocktails or in SDS Laemmli sample buffer. Cell lysates (RIPA buffer) were quantified for proteins content with the Bio-Rad DC Protein Assay kit. Proteins (25-50 μg) were then separated by 8-12% SDS-PAGE and transferred to nitrocellulose membranes 0.45 μm (GE Healthcare Life Sciences). The membranes were blocked with 5% non-fat milk (EuroClone) or 2% BSA (Sigma-Aldrich) in 1x TBS pH 7.6-8.0 containing 0.1 or 0.2% Tween 20 (TBST) for 2 hour at room temperature (RT) and subsequently probed with primary antibodies in 5% non-fat milk or 2-5% BSA in TBST overnight at 4°C. Then membranes were washed 10 minutes for 3 times with TBST and probed with horseradish peroxidase-conjugated secondary antibodies in 5% non-fat milk in TBST for 1 hour at RT. Chemidoc XRS Bio-Rad was used for images acquisition with a chemi-luminescent camera, band signals were quantified using ImageLab 4.0 Bio-Rad software.

Equal amounts of protein were separated by SDS-PAGE in 4–12% Bis-Tris gels (Invitrogen), and transferred to nitrocellulose membranes (0.45 μm, GE healthcare, Little Chalfont, UK). The filters were blocked in TBS-T containing 5% non-fat dried milk for 1 h at room temperature or overnight at 4°C. Proteins were visualized using appropriate primary antibodies. All primary antibodies were diluted in TBS-T and incubated with the nitrocellulose blot overnight at 4°C. Incubation with secondary peroxidase coupled anti-mouse, anti-rabbit or anti-goat antibodies was performed by using the ECL system (Thermo Scientific SuperSignal West Pico, 34080; Amersham ECL Prime, RPN2232). Protein loading was monitored by normalization to β-actin. Blots were scanned and quantitative densitometric analysis was performed by using ImageJ (http://imagej.nih.gov/ij/).

Equal amounts of protein were separated by SDS-PAGE in 4–12% Bis-Tris gels (Invitrogen), transferred to nitrocellulose membranes (0.45 μm, GE healthcare, Little Chalfont, United Kingdom) and blocked in PBS-T containing 5% non-fat dried milk for 1 h at room temperature. The nitrocellulose filters with 0.1 μM pores followed by cross-linking treatment with PFA 0.4% were used to increase the retention of low-molecular-weight monoubiquitin after transfer and before its detection (Emmerich and Cohen, 2015 (link)). Proteins were visualized using appropriate primary antibodies. All primary antibodies were diluted in PBS-T and incubated with the nitrocellulose membrane overnight at 4°C. Incubation with secondary peroxidase coupled anti-mouse or anti-rabbit antibodies was performed by using the ECL system (Thermo Scientific SuperSignal West Pico, 34080; Amersham ECL Prime, RPN2232). Protein loading was monitored by normalization to β-actin. The films were digitized using a professional scanner and quantitative densitometric analysis (expressed in arbitrary units normalized on the expression of the housekeeping protein β-actin) was performed by using ImageJ (see footnote 1).