Glut5

Frozen segments of jejunum were prepared for Western blot assessments using similar Western blot methodology as previously described [35 ,37 (link),38 (link)]. Protein Assays were performed to determine homogenate concentration. Blots were incubated with the following primary antibodies overnight at 4 °C: SGLT-1 (1:500, Abcam Cambridge, UK), GLUT2 (1:500, Cell Signaling, Danvers, MA, USA), GLUT5 (1:3000, Cell Signaling, Danvers, MA, USA), Tau 5 (1:750, Calbiochem, Millipore-Sigma, Burlington, MA, USA), CDK5 (1:750, Cell Signaling, Danvers, MA, USA), Caspase-3 (1:1000, Cell Signaling, Danvers, MA, USA), Alpha actin smooth muscle (1:750, Invitrogen, Thermo-Fisher, Waltham, MA, USA). To re-probe for GAPDH, blots were incubated with anti-GAPDH primary antibody (1:4000, Thermo Scientific, Rockford, IL, USA) or anti-actin (1:3000, EMD Millipore, Billerica, MA, USA) for 1 h at room temperature. Blots were washed and then incubated with the appropriate secondary antibody anti-mouse IgG (H + L) (1:15,000, Dylight, Thermo Scientific, Rockford, IL, USA), and anti-rabbit IgG (H + L) Dylight (1:15,000, Thermo Scientific, Rockford, IL, USA), for 1 h at room temperature. Images of membranes were taken with all proteins of interest normalized to either actin or GAPDH. Band density was analyzed using Odyssey-Clx (LI-COR, Lincoln, NE, USA) and Image Studio (LI-COR, Lincoln, NE, USA).