Odyssey cls infrared imager system

Protein extracts were prepared from human left ventricles as decribed[23 (link)]. The total lane density of transferred proteins stained with Ponceau S was used to control for loading/transfer differences. Primary antibodies used were as follows: SOX4 (Diagenode, CS129100), BCL6B(Abgent, AP20369c), SMYD1 (Abcam, ab32489), CELEC3B ( Abcam, . ab108999), SERPINE1 (Abcam, ab66705) and SLC27A3 (Thermo Fisher Scientific 12943–1-AP). Secondary antibodies coupled to Alexa Fluor 680 (Invitrogen Molecular Probes) or IRDye 800 (LI-COR Biosciences) were used, and the Odyssey CLS infrared imager system (LI-COR Biosciences) was used for visualization of Western blot signals. Odyssey version 1.2 imaging software was used to process all images.

Protein extracts were prepared from human left ventricles (LV) from the same cohorts of subjects in MAGNet as previously described ¹³ . Briefly, 60 mg of LV were homogenized using a tissue homogenizer with a 5×75mm Flat Bottom Stainless Steel generator probe (OMNI international, THP115) in a RIPA buffer (ThermoFisher, 89900) containing protease and phosphatase inhibitors (ThermoFisher, 78410 & 78420). Thirty µg total proteins were separated in SDS-polyacrylamide gel electrophoresis, transfer to nitrocellulose membranes or Polyvinylidene fluoride or polyvinylidene difluoride (PVDF), and blotted with primary antibodies overnight at 4°C. The total lane density of transferred proteins stained with Ponceau S was used to control for loading/transfer differences. Primary antibodies used were as follows: SOX4 (Diagenode, CS129100), SOX8 (GeneTex, GTX129949), SOX9 (Millipore, AB5535), SOX15 (ThermoFisher, 25415–1-AP). Secondary antibodies coupled to Alexa Fluor 680 (Invitrogen Molecular Probes) or IRDye 800 (LI-COR Biosciences) were used, and the Odyssey CLS infrared imager system (LI-COR Biosciences) was used for visualization of Western blot signals. Odyssey version 1.2 imaging software was used to process all images.