Irdye 680cw labeled secondary antibodies

Immunoblot analyses were performed as previously described (Joshi et al., 2014). Briefly, proteins were extracted in enzyme immunoassay precipitation buffer containing 250 mM Tris base, 750 mM NaCl, 5% NP‐40, 25 mM EDTA, 2.5% sodium deoxycholate, 0.5% sodium dodecyl sulfate and an EDTA‐free protease and phosphatase inhibitors cocktail tablet (Roche Applied Science, Indianapolis, IN, USA), sonicated, centrifuged at 125,000 g for 45 min at 4°C, and supernatants used for immunoblot analysis. Total protein concentration was determined by using BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Samples were electrophoretically separated using 10% Bis–Tris gels or 3%–8% Tris–acetate gel (Bio‐Rad, Richmond, CA, USA), according to the molecular weight of the target molecule, and then transferred onto nitrocellulose membranes (Bio‐Rad). They were blocked with Odyssey blocking buffer for 1 hr and then incubated with primary antibodies overnight at 4°C. After three washing cycles with T‐TBS, membranes were incubated with IRDye 800CW or IRDye 680CW‐labeled secondary antibodies (LI‐COR Bioscience, NE, USA) at 22°C for 1 hr. Signals were developed with Odyssey Infrared Imaging Systems (LI‐COR Bioscience). Actin and GAPDH were used as internal loading controls. Primary antibodies used in this paper are summarized in Table 2.