Tris hcl precast gel

Duodenum segments were rinsed in ice cold Phosphate Buffered Saline (PBS) + 5 mmol/L EGTA (pH 7.42) and mucosal scrapings were prepared as described by Wang et al.^{(25 (link))} Total protein concentration was measured with a Detergent Compatible Protein Assay (BioRad Laboratories, Hercules CA) according to the manufacturer’s instructions and Western Blot analysis was conducted as described elsewhere.^{(26 )} Samples from individual mice and a pooled tissue sample for each genotype were analyzed. Briefly, 30 µg of protein were separated onto a 12.5% Tris-HCL precast Gel (BioRad Laboratories, Hercules CA) and transferred onto a 0.45 µm PVDF membrane (EMD Millipore, Billerica, MA). For the primary antibodies the membrane was incubated with anti-VDR (1:500 dilution, D-6 Santa Cruz Biotech, Dallas, TX)^{(25 (link))}, or anti-βactin (1:1000 dilution, Sigma-Aldrich, St. Louis, MO). The membrane was then incubated with the HRP-conjugated goat anti-mouse IgG light chain, secondary antibody (1:5000 dilution; Jackson ImmunoResearch Laboratories Inc., West Grove, PA). The HRP signal was detected with the ECL Super Signal system (ThermoScientific, Rockford, IL). The relative band densities were analyzed using Image J 1.48v software (NIH).

N2a cells transiently transfected with plasmids were gently scrapped in cold PBS and collected by centrifugation at 12,000 rpm for 1 min at 4 °C. Cell pellet was lysed in RIPA buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) (Sigma-Aldrich) containing protease inhibitors (Complete mini, EDTA-free, Roche) and centrifuged at 12,000 rpm for 20 min at 4 °C. Protein concentration of the supernatant was measured by the BCA method (Thermo Scientific). Supernatant (50 μg) was loaded onto a Tris-HCl precast gel (Bio-Rad) and run with MOPS buffer. The gel was transferred to a PVDF membrane and incubated with RU-369 rabbit antibody (1:1,000 dilution) developed in house to detect the C-terminus of APP^{36 (link)}.

Cell pellets were resuspended in Lysis Buffer (Promega) followed by freeze/thaw in dry ice/ethanol bath to obtain the whole lysate. The cell lysates were mixed with SDS sample buffer (Invitrogen) and 2% β-mercaptoethanol, boiled for 5 min and resolved by SDS-PAGE using 10–20% Tris/HCl precast gel (Bio-Rad). Precision plus protein standards (Bio-Rad) were included as references. After gel electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membrane for further probing with antibodies against proteins of interest. Western Lightning Plus ECL (PerkinElmer) or ECL™ Prime Western Blotting Detection Reagent (Fisher Scientific) were used for visualization of protein immunoreactivities. Immunoreactivity of β-actin was used as loading controls.