Semi dry turbo blotter system

Quantification of mRNA and protein levels was performed by RT-qPCR and western blotting respectively, as described previously [24 (link)]. Total RNA was extracted using Ambion Pure-Link kits (Thermo Fisher) and was reverse transcribed using QuantiNova RT kit (Qiagen) and random primers. Quantitative PCR was performed using Roche SYBR Green using a Qiagen Roto-Gene Q PCR machine (20’@95 °C;20’@62 °C;20’@72 °C). Primers sequences are described in Supplement Table 1. Data were normalised to total amount of RNA. Western blots were performed using a Mini-Protean II system. Proteins were transferred to PVDF membrane using a semi-dry Turbo blotter system (Bio-Rad) and detected using ECL and a digital ChemiDoc imaging system (Bio-Rad). Phos-tag gels were prepared containing 100 μM Phos-tag acrylamide and 20 μM MnCl₂ according to the manufacturer's instructions (Alpha Laboratories).

Quantification of mRNA and protein levels was performed by qRT-PCR and Western blotting respectively, essentially as described previously [16] (link). Total RNA, extracted using Ambion Pure-Link kits and was reverse transcribed using QuantiTect RT kit (Qiagen) and random primers. Quantitative PCR was performed using Roche SYBR Green using a BioRad Roto-Gene Q PCR machine (20′@95 °C; 20′@62 °C; 20′@72 °C). Primers sequences are described in supplement table 1. Data was normalised to total RNA levels in each reaction. Primers sequences are detailed in Table 1. Western blots were performed using a Mini-Protean II system. Proteins were transferred to PVDF membrane using a semi-dry Turbo blotter system (Bio-Rad) and detected using ECL and a digital ChemiDoc imaging system (Bio-Rad). Phos-tag gels were prepared containing 100 μM Phos-tag acrylamide and 20 μM MnCl₂ according to the manufacturer's instructions (Alpha Laboratories).

Total cell lysates were prepared in 1x reducing Laemmli sample buffer (2% SDS, 10 glycerol, 50 mM Tris pH 6.8, 2.5% β-mercaptoethanol, 0.002% bromophenol blue). Proteins were denatured by heating to 100 °C for 4 min prior to electrophoresis. Electrophoresis was performed using Bio-Rad 4–15% polyacrylamide mini-TGX gels in a Mini-Protean II system. Proteins were transferred to PVDF membrane using a semi-dry Turbo blotter system (Bio-Rad). Membranes were blocked for 1 h at room temperature in 5% low-fat milk powder in Tris buffered saline containing 0.2% Tween20 (1TBS.T) before incubation with primary antibody overnight at 4 °C. Following extensive washing in TBS.T, blots were incubated with HRP-conjugated secondary antibodies. Specific proteins were detected using Immobilon ECL reagent and a ChemiDoc-MP digital imaging system (Bio-Rad, Watford, U.K.).