Ab50533

Protein lysates from whole pancreas were prepared in 1 mL of RIPA buffer supplemented with phosSTOP phosphatase and protease inhibitors (Roche). Protein concentrations were measured using the BCA kit (Thermo Scientific) and then diluted in 4 x Laemmeli buffer (40% glycerol, 8% SDS, 240 mM Tris-HCl pH 6.8, 5% b-mercaptoethanol, 12.5 mM EDTA, 0.04% bromophenol blue) and RIPA buffer at 10 mg/mL. 10 mL of lysate was resolved on 8% SDS-PAGE and transferred to Nitrocellulose membrane (BioRad). Membranes were blocked with 5% milk in Tris-buffered saline with 1% Tween 20 (TBST) for 1hr, followed by overnight incubation at 4 C with primary antibodies such as NFIA (Fancy et al., 2012 ; at 1:1000), Chga (DSHB at 1:200), Sox9 (Santa Cruz Biotech. SC20095 at 1:200), Chmp4c (Abcam, ab155668 at 1:500), Mib1 (Novus, NBP1-85210 at 1:500), Dll1 (from Christel Brou at 1:1000), Notch1 (from Christel Brou at 1:1000), NICD1 (Abcam, ab8925 at 1:1000), Hes1 (VWR, 100192-118 at 1:500), Rab5 (Abcam, ab18211 at 1:200), Rab7 (Abcam, ab50533 at 1:200), and b-actin (Sigma, A5441 at 1:5000). Membranes were washed three times with TBST before incubation for 1 hour with either anti-rabbit IgG conjugated to horseradish peroxidase (HRP) for NFIA (GE Life Sciences) or anti-mouse IgG-HRP for b-actin (GE Life Sciences). Expression was quantified using ImageJ and normalized to b-actin.