HMGA1 Protein Analysis via SDS-PAGE and Western Blot

SDS PAGE and western blot analyses were performed essentially as previously described [19 (link)]. WB signal intensities were normalized to total proteins using densitometric analysis of Ponceau S stained membranes. Primary antibodies used were: α-HMGA1 (homemade) and α-H1.2 (ab181977, Abcam, Cambridge, UK). Secondary antibodies were: α-Rabbit IgG Peroxidase conjugate (#A0545, Sigma Aldrich, St. Louis, MO, USA) and α-Mouse IgG Peroxidase conjugate (#A9044, Sigma Aldrich, St. Louis, MO, USA). The α-HMGA1 antibody was produced by immunization of a rabbit with a recombinant HMGA1a protein lacking the C-terminal acidic domain. α-HMGA1 antibodies were affinity purified form serum using a resin (Affi-Gel 10 Gel #153-6046, Bio-Rad Laboratories, Hercules, CA, USA) covalently derivatized with HMGA1 recombinant protein. Recombinant proteins used for immunization and affinity purification were purified by reversed-phase HPLC and checked by mass spectrometry. Antibodies were eluted with a Glycine 0.2 M pH 2.8 solution and acidic pH was neutralized with a Tris 2 M pH 8 solution. Antibodies were stored at −20 °C in small aliquots.