Sglt1

Tissues from rats were fixed using 10% buffered formalin, dehydrated, embedded in paraffin, and sectioned into 5 μm-thick sections. Hematoxylin and eosin (HE) staining was used to assess cardiac injury, whereas Masson’s trichrome staining was used to detect collagen fibers, and the slides were observed under an optical microscope. For immunohistochemistry, sections were stained with a primary antibody against SGLT1 (1:200, Abcam) and then stained with a secondary antibody. After washing with PBS, the slides were incubated with 3,3′-diaminobenzidine. The detailed procedure has been described previously (Lin et al., 2019 (link)). Semiquantitative analysis was performed using image analysis software (Image-Pro Plus, Media Cybernetics).

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).