Hyperfilm ecl

Ventral midbrain and dorsal midbrain (DM) tissues from E12.5 CD-1 mouse embryos were dissected in ice-cold PBS and homogenized in RIPA buffer [50 mM Tris–HCl pH7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, 3 mM EDTA, and protease inhibitors (complete Mini; Merck)]. Total proteins (20 μg per sample, determined with Pierce BCA Protein Assay; Thermo Fisher Scientific) were separated by SDS-PAGE, blotted on Hybond-LFP membranes (Merck) and stripped as described by Fischer et al. (2011) (link). Blots were probed with two different rabbit anti-LEF1 mAbs [C12A5 (#2230) and C18A7 (#2286); 1:1000, Cell Signaling Technology], mouse anti-b-actin (ACTB) mAb (1:5000; abcam ab6276) and HRP-conjugated goat-anti-rabbit (Dianova 111-035-003) and goat-anti-mouse (Dianova 115-035-003) secondary antibodies (each 1:5000), developed in ECL substrate and exposed to Hyperfilm ECL (Merck).

The method is based on the presence/absence of 43 DNA spacer sequences, which are interspersed between 36 conserved loci located in the Direct Repeat (DR) region (37 (link), 40 (link)). Spoligotyping was performed according to Kamerbeek et al. (41 (link)): PCR amplification of DR loci was performed using DRa and DRb primers and products were biotinylated and hybridized to a membrane containing oligonucleotides for each spacer sequence. After hybridization, the membrane is washed and then incubated in diluted streptavidin-peroxidase conjugate (Roche, United States). Membrane was exposed to chemiluminescent Amersham ECL reagents (GE Healthcare, United Kingdom) and located in an X-Ray cassette on a Hyperfilm ECL (Merck, United States). After the reaction, the film was inserted into a film developer solution in a dark room after which it is moved to the fixer solution. Thereafter, the film is dried and ready for the interpretation of the result. After developing the film a positive/negative signal is recorded in binary or octal formats for genotyping interpretations (Supplementary Data 1). The results are compared against the Fourth International Spoligotyping Database (SpolDB4) (42 (link)).

Cells used for immunoblotting were cultured in T25 flasks and when confluent, washed in PBS and then lysed in situ with 200 μl of mammalian protein extraction reagent (M-PER, Pierce, Loughborough, UK) containing complete protease inhibitor tablets (Roche) for 30 min at room temperature. Protein levels in the lysates were assessed using a BCA assay (Pierce). Cell proteins (typically 30 μg protein per lane) were separated on 12.5%, 0.75 mm thick polyacrylamide SDS gels and electrophoretically transferred to 0.2 μm PVDF membranes (BioRad). The blots were probed with primary antibodies (1 : 1000, cell signaling) raised against AMPKα, phospho-AMPKα, Acetyl-CoA Carboxylase (ACC), Phospho-acetyl-CoA Carboxylase (phospho-ACC) at room temperature for 3 h or HO-1 (1 : 200, Santa Cruz) at 4 °C overnight. Next, membranes were washed with PBS for 30 min prior to incubating with the appropriate anti-rabbit or anti-mouse horse radish peroxidise-conjugated secondary antibody (1 : 2000; Amersham Pharmacia Biotech, Buckinghamshire, UK) for 1 h at room temperature. Following this incubation, membranes were washed in PBS for 30 min and bands visualized using an enhanced chemiluminescence detection system and hyperfilm ECL (Merck, UK).