Western Blot Protocol for Protein Analysis

Protein lysates were prepared using Totex lysis buffer, as described before [31 (link)], loaded on an SDS-PAGE gel, and blotted onto nitrocellulose membranes (Bio-Rad). The list of primary antibodies can be found in Supplementary Table S3. Note that the primary MR antibody is of a non-commercial source, and hence, different batches were used throughout the course of this research (clone 6G1, kind gift Dr. Gomez-Sanchez). As secondary antibodies, we used species-specific HRP-conjugated antibodies (cat nr: NA931, NA934, GE-Healthcare). To visualize results, Pierce ECL (Plus) (Thermo Fisher Scientific), Westernbright Quantum or Sirus (Isogen), or ECL Prime (GE Healthcare) served as chemiluminescent substrates, and signals were developed using X-ray films or imaged on a ProXima 2850 (Isogen) or Amersham 680 (GE healthcare) imaging system. Band densitometric analyses were performed using ImageJ.