Ab38548

For the determination of transcription factor protein abundance, protein components of lysates (100 µg) were separated by SDS-polyacrylamide gel electrophoresis on 12% (w/v) polyacrylamide mini gels, containing 0.1% (w/v) SDS, whilst for the determination of SGLT1 protein abundance, protein contents of BBMV (20 µg) were separated using 8% (w/v) polyacrylamide mini gels. Membranes were incubated with either our custom-made antibody to SGLT1^{40 (link), 41 (link)} or with antibodies to Neurogenin 3 (ab38548, Abcam, Cambridge, UK), NeuroD1 (ab109224), HATH1 (ab168374) and Hes1 (ab108937). Immunoreactive bands were detected by affinity purified horseradish peroxidase-linked anti-rabbit secondary antibody (DAKO Ltd, Cambridge, UK) and visualised using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Hertfordshire, UK) and Bio-Max Light Chemiluminescence Film (Sigma-Aldrich, Poole, Dorset, UK). The intensity of the immunoreactive bands was quantified using scanning densitometry (Total Lab, Newcastle-upon-Tyne, UK). β-actin protein abundance was used as a loading control.

Westerns were performed and normalized against GAPDH (Glyceraldehyde 3-phosphate dehydrogenase, 37 kd, 1:10,000, MAB374, Millipore, Billerica, MA, USA) as previously reported by our group [32 (link)]. For detection of pAMPK, NaF (50 mM) was added to all buffers, which had been demonstrated to be an effective phosphoseryl and phosphothreonyl protein phosphatase inhibitor [33 (link)]. Antibodies were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA) unless otherwise specified: AgRP (1:500, 14 kd, sc-50299); POMC (1:500, 30 kd, sc-20148); Hes1 (1:500, 35 kd, sc-25392); Mash1 (1:500, 30 kd, sc-13222); Ngn3 (1:1000, 23 kd, ab38548, Abcam, Cambridge, MA); and AMPK (1:1000, 63 kd, sc-19128). All values were normalized to GAPDH and presented as fold change.