Odyssey imager system

Protein samples were boiled for 5 min in Laemmli loading buffer (except PAD1 samples, that remained unboiled), run in a 10 or 12% SDS/PAGE and transferred to a nitrocellulose membrane. After blocking with 5% milk in PBS-T (0.05% Tween in PBS) for at least 30 min at room temperature, membranes were incubated with antibodies overnight at 4°C under agitation in 5% milk in PBS-T. The primary antibodies were used at the following dilutions: αPAD1 (1:1000); αNeo (1:1000, Neomycin Phosphotransferase II, Merck-Millipore); αEF1α (1:7000, Elongation Factor 1-alpha, Merck-Millipore); αREG9.1 (1:2500; polyclonal antibody generated by Eurogentec against the recombinant protein), αBB2 (1:5,[51 (link)]). After 3 washes in PBS-T, two different systems for antibody detection were used. Chemiluminescence required 1h incubation at room temperature with a horseradish peroxidase-conjugated secondary antibody in 5% milk (in PBS-T) that was either α-rabbit (1:5000) or α-mouse (1:5000). The membrane was then washed three times in PBS-T and revealed using Pierce ECL2 western blotting substrate (ThermoScientific). Alternatively, protein was visualised by incubating the membranes for 1h at room temperature with a secondary antibody conjugated to a fluorescent dye diluted 1:5000 in 50%Odyssey Blocking buffer/50% PBS-T and finally scanning the membrane with a LI-COR Odyssey imager system.

Protein samples were boiled for 5 min in Laemmli loading buffer (except PAD1 samples, that remained unboiled), separated by SDS–PAGE (NuPAGE gel 4–12% Bis-Tris, Invitrogen) and visualized either by Coomassie staining or SYPRO Ruby Protein Gel Stain (Invitrogen) using a Typhoon 9400 scanner (Amersham Biosciences) with lex = 457 nm and lem = 610 nm. Alternatively, proteins were separated by SDS–PAGE on NuPAGE 4–12% Bis-Tris gels and blotted onto polyvinylidene difluoride (PVDF) membranes (Pierce). After blocking with Odyssey Blocking buffer for at least 30 min at room temperature (RT), membranes were incubated with antibodies 1- to 3 hr at RT or overnight at 4°C under agitation in 2% BSA in TBS-T (0.1% Tween in TBS). The primary antibodies were used at the following dilutions: αPAD1 (1:1000); αBB2 (1:5, [Bastin et al., 1996 (link)]); αThioP (1:1000, Thio-phosphate ester [51-8], Abcam). After three washes in TBS-T, proteins were visualised by incubating the membrane for 1 hr at RT with a secondary antibody conjugated to a fluorescent dye diluted 1:5000 in 50%Odyssey Blocking buffer/50% TBS-T. Finally, membranes were scanned using a LI-COR Odyssey imager system.