Quantification of mRNA and Protein Levels

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).