Storm device

PCR products were submitted to TTGE for sequence-specific separation using the DCode Universal Mutation Detection System (Bio-Rad, Paris, France), a 1 mm-thick, 16 × 16 polyacrylamide gel (as described by [42 (link)]) and 7 L of 1.25× Tris-acetate-EDTA electrophoresis buffer. The electrophoresis was performed at a fixed voltage of 63 V for 15 h, with an initial temperature of 66 °C and a ramp rate of 0.3 °C/h. The gel was stained in the dark by immersion for 30 min in a solution of SYBR Gold^® Nucleic Acid Gel Stain (Invitrogen, Eugene, OR, USA) and was then read on a Storm device (Molecular Dynamics, Bondoufle, France). The TTGE profiles were analyzed with GelCompar software (version 2.0, Applied Maths, Kortrijk, Belgium) in order to determine (i) the number of bands in each lane; and (ii) each band’s position and intensity. A principal component analysis (PCA) was used to identify patterns in data and to highlight similarities and differences. The degree of similarity between patterns was measured by calculating Pearson’s coefficient.

BV-2 cells were washed twice with ice-cold PBS at 4°C. Cells were lysed in radioimmunoprecipitation assay buffer [RIPA, in mM: 50 Tris HCl, pH 7.4; 150 NaCl; 5 EDTA; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate (SDS)] supplemented with complete mini protease inhibitor cocktail tablets and 1 mM of dithiothreitol (DTT) at 4°C. Samples were denatured by adding 6x concentrated sample buffer (0.5 M Tris, 30% glycerol, 10% SDS, 0.6 M DTT, 0.012% bromophenol blue) and heating for 5 min at 95°C. When blotting for CD39, cells were lysed in non-reduced conditions.
Samples were separated in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% milk and then were incubated with the antibodies indicated in Table 1. Immunoreactive bands were visualized using Enhanced Chemi-Fluorescence system (ECF; GE Healthcare) on a Storm device (Molecular Dynamics, GE Healthcare) or with Enhance Chemiluminescence system (ECL; Bio-Rad) on a Versadoc (Bio-Rad). Digital quantification of band intensity was performed using Quantity One software (Bio-Rad). The membranes were reprobed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to control for similar amounts of protein.