Odyssey infrared image detecting system

Frozen kidney samples were homogenized in 0.5 ml lysis buffer and processed for Western blotting as previously described (Ma et al., 2014 (link)). Blots were probed for phospho-c-Jun and α-tubulin as the loading control using goat anti-rabbit Alexa Fluor 680 or donkey anti-mouse IRDye 800 secondary antibodies (Molecular Probes) and detected using the Odyssey Infrared Image Detecting System (LICOR). Densitometry analysis used ImageJ software, NIH.

Frozen kidney samples were homogenized in 0.5 mL of lysis buffer as previously described [46 (link)]. Samples were then sonicated, incubated at 4 °C for 60 min, centrifuged, and the supernatant heated at 95 °C for 5 min in SDS-PAGE sample buffer. Samples were separated on 4–20% gradient gels and transferred to nitrocellulose membranes. Membranes were incubated with Odyssey Blocking Buffer (LI-COR, Lincoln, NE, USA), incubated with primary antibodies in overnight at 4 °C, washed, incubated with goat anti-rabbit Alexa Fluor 680 or donkey anti-mouse IRDye 800 for 1 h, and scanned with the Odyssey Infrared Image Detecting System (LI-COR). Densitometry analysis was performed by the Gel Pro analyzer program (Media Cybernetics, Rockville, Maryland USA).