Protein quantification kit

Proteins from PBMCs were isolated with the NucleoSpin RNA/Protein Kit (Macherey-Nagel). Nuclear and cytoplasmic proteins from transfected HEK293T cells were obtained with the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Concentrations were measured with Protein Quantification Kit (Macherey-Nagel).

Leaf discs were ground in dry ice and protein extractions performed as described in Lopez‐Calcagno et al. (2017) and using the nucleospin RNA/Protein kit (Macherey‐Nagel, http://www.mn-net.com/). Protein quantification was performed using the protein quantification Kit from Macherey‐Nagel. Samples were loaded on an equal protein basis (8–0.1 μg per well depending on the experiment), separated using 12% (w/v) SDS‐PAGE, transferred to nitrocellulose membranes, and probed using antibodies raised against each of the GDC complex proteins (Timm et al., 2012) and LA (Merck KGaA, Darmstadt, Germany). Proteins of interest were detected using horseradish peroxidase conjugated to the secondary antibody and ECL chemiluminescence detection reagent (Amersham, Buckinghamshire, UK). In addition to the aforementioned antibodies, samples were probed using antibodies raised against transketolase (Henkes et al., 2001) and actin (Agrisera, via Newmarket Scientific, UK).