Ecl prime chemiluminescence detection kit

The imidazo-[1,2-b]-pyridazine-based Pim1 kinase inhibitors were synthesised in the Knapp Laboratory and stored at a concentration of 50 mM in DMSO. The ABCG2-specific inhibitor fumitremorgin C (FTC), doxorubicin, topotecan and mitoxantrone were purchased from Sigma-Aldrich and prepared as 50 mM aliquots in DMSO. mitoxantrone (Tocris) was prepared at 50 mM in DMSO, and flavopiridol (Biomol International) was prepared at 10 mM in DMSO. All compounds were stored at -20 °C. [ 3 H]-mitoxantrone (148 GBq/mmol) was purchased from Moravek Biochemicals (Brea, USA). The anti-ABCG2 (BXP-21) and the anti-Pim antibody (12H8) were purchased from Santa Cruz Biotechnology and the goat anti-mouse horseradish peroxidase-conjugated antibody from DAKO. The ECL Prime chemi-luminescence detection kit was purchased from GE. All plasticware was purchased from either Fisher Scientific UK or Greiner Bio-One.

Proteins separated in polyacrylamide gels were transferred onto nitrocellulose membranes using a Semi-Dry Transfer system (Bio-Rad, Hercules, CA). The transfer of proteins was carried out according to Schertl et al. (2014) at 12 V (max) and 400 mA for about 90 min using transfer buffer (25 mM Tris, 192 mM glycine, 1.3 mM SDS, 20% Methanol). The blots were rinsed several times in TTBS buffer [0.1 M Tris-HCl pH 7.5, 0.155 M NaCl and 0.01% (v/v) Tween 20] to remove all methanol and then incubated with 1:800 primary antibody diluted in TTBS buffer for about 16 h at room temperature. Antibodies for OXPHOS subunits are described in Peters et al. (2012) which are the 51 kDa subunit (complex I), SDH1-1 (complex II), alpha-MPP (complex III), COX2 (complex IV) and the ß-subunit V). The IVDH antibody was described by Gu et al. (2010) . After antibody incubation, membranes were incubated for 2 h with a 1:5000 diluted secondary antibody directly coupled to horseradish peroxidase. All blots were developed using a homebrew protocol luminol chemical-based as following described [Tris 100 mM pH 8.5; p-cumaric acid (15% v/v); luminol (44% v/v); H 2 O 2 (15% v/v)] the ECL prime chemiluminescence detection kit from GE Healthcare (Munich, Germany).