Nupage mini gel

For whole cell lysates, PBS washed cells were lysed in RIPA Buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1x phosphatase (Phos-Stop, Roche) and protease (Complete, EDTA-free, Roche) inhibitor mixes) on ice for 20 min. Lysates were sonicated with a probe at medium intensity for 5 s in a Soniprep 150 instrument and clarified by centrifugation at 13000 g for 15 min at 4°C. Protein concentration was quantified using the DC Protein Assay (Bio-Rad) according to the manufacturer’s instructions. Proteins were denatured in 2X NuPAGE LDS sample buffer (Invitrogen) and 1% 2-metcaptoethanol (Sigma-Aldrich) for 5 min at 95°C. Proteins were separated by SDS-PAGE using NuPAGE mini gels (Invitrogen) and transferred onto 0.2 μm pore Nitrocellulose membrane (Amersham Protran; Sigma-Aldrich). Membranes were blocked with 5% skim milk/TBST (TBS/0.1%Tween-20) for 1 h at room temperature and probed with the indicated primary antibodies overnight at 4°C. Membranes were then washed 3 times for 10 min with TBST, incubated with appropriate secondary antibodies conjugated to a horseradish peroxidase (HRP) for 1 h at room temperature and washed again 3 times for 10 min with TBST. Immunoblots were developed using Clarity or Clarity Max Western ECL Substrate (Bio-Rad).

All SDS-PAGE gels used were Invitrogen (Paisley, UK) 15 or 10 well 4-to-12% bis tris precast Nu-PAGE mini gels and were run according to the manufacturer's instructions. Invitrogen's Mark12™ unstained standard was used as a molecular weight marker at a 1 in 20 dilution. After running at 200 V for 35 min the gels were silver stained with the SilverSnap 2 stain kit from Pierce (Northumberland, UK). Reduction of samples was performed with DTT. Gels were photographed on a MEDALight light panel (Morco, UK).

Proteins were separated by SDS-PAGE using NuPAGE mini gels (Invitrogen) and transferred onto a PVDF membrane (Millipore, Immobilon-P) using standard procedures. After transfer, the membrane was blocked in 5% skim milk/ TBST (TBS/ 0.1% Tween-20) for 30 min at room temperature and incubated with the indicated primary antibody (diluted in 5% skim milk/ TBST) for overnight at 4°C. The membrane was then washed 5 times for 5 min with TBST, incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, and washed again 5 times for 5 min with TBST. The immunoblot was developed using ECL Western Blotting Reagent (Sigma) or SuperSignal West Femto (Thermo Fisher Scientific). All incubations were carried out on a horizontal shaker.