Power blotter

Tissue samples were homogenized in 50 mM MOPS, pH = 7.0, 20% glycerol and 2.7 mM EDTA in IKA T10 basic homogenizer. Sonication and detergent extraction of the homogenates was performed as previously described for the rat brain homogenates^{68 (link)}. For Western blotting, the non-sonicated homogenates were diluted to app. 8.5 mg tissue per mL, and proteins were separated on hand-cast 10% acrylamide gels containing 0.5% 2,2,2-trichloroethanol. For detection of the DHTKD1-encoded protein, gels were blotted onto PVDF membrane using PowerBlotter (Thermo Scientific) and probed with anti-DHTKD1 antibodies (PA5–24208, Invitrogen, 1:1000) and HRP-conjugated secondary antibodies (P-GAM Iss, Imtek, Russia, 1:1000) in iBind Western Device (Thermo Scientific). Proteins were visualized using Clarity Western ECL substrate (Bio-Rad) and ChemiDoc MP Imaging System (Bio-Rad). Band intensities were calculated using ImageLab software (Bio-Rad) and normalized by relative whole protein level, measured as combined signal in the 20–250 kDa region of the corresponding gel lane using stain-free gel mode in ChemiDoc MP and ImageLab. Raw images of anti-DHTKD1-stained membrane and of total protein visualized in gel are given in Fig. S1A,B, respectively.

Equal amounts (10 µg) of protein corresponding to samples of the membrane-enriched fraction were prepared in Laemmli SDS-PAGE loading buffer, and denatured for 5 min at 95 °C. Samples were loaded onto 4–12% NuPAGE Bis-Tris gels, and separated at 180 V for 1 h. PageRuler Plus pre-stained protein ladder (Thermo Fisher Scientific) was used as the molecular weight marker. Electrophoretic transfer was achieved using a Power Blotter (Thermo Fisher Scientific) onto nitrocellulose membrane, which was subsequently blocked for 1 h using 5% non-fat dried milk powder. Development of blots was carried out using chemiluminescence in a dark room. Western blots for membrane protein enrichment analysis were probed with the following primary antibodies: anti-caveolin-1 (Ab2910, Abcam) and anti-sodium potassium ATPase (Ab76020, Abcam). Western blot validation of CD55 expression was carried out using anti-CD55 (Ab133684, Abcam). Coomassie blue-stained gels corresponding to the Western blot of CD55 are shown in Supplementary Figure S3.

Lysates were prepared using NP-40 lysis buffer supplemented with a protease-phosphatase inhibitor cocktail (IBI Scientific). Lysates were ran on a 10% SDS-PAGE gel prior to transferring to nitrocellulose blots using semi-dry transfer system (PowerBlotter; Thermo Fisher). Membranes were stained with primary antibody prior to wash and secondary antibody labeling. Protein bands within blots were then detected using the Licor Odyssey imaging system (Licor, Lincoln NB) and analyzed using Image Studio 5.2 software (Licor).

SCL/LMO1 parental 7298 cells and cytarabine resistant 7298 cells were lysed in RIPA lysis buffer (sc-24948A, Santa Cruz) with 1 mM sodium orthovanadate, 1 mM PMSF and protease inhibitor cocktail on ice for 15 min. The protein concentration of sample is determined by Micro BCA^™ Protein Assay Kit (Cat#23235, Thermo Fisher Scientific). The protein sample is prepared with Laemmli buffer (Cat#1610747, BIO-RAD) to a final concentration of 1 ug/ul. 20 μl protein samples were applied to 10% SDS-PAGE gels, separated by electrophoresis at 80 V for 30 min and 120 V for 90 min. After electrophoresis, protein was transferred to a nitrocellulose membrane using Semi-dry Transfer System (Power Blotter, Thermo Fisher Scientific). The membrane was then blocked in 5% non-fat dry milk (Blotting-Grade Blocker, BIO-RAD) in TBST (10 mM Tris–Cl, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 h. The membrane was then incubated with primary antibody overnight at 4°C followed by incubation with a secondary antibody conjugated to horseradish peroxidase (HRP), and visualized using a chemiluminescence kit (GE Healthcare, RPN2106). Specific antibodies to DCK (Cat# ab186128, Abcam), β-Actin (Cat# A1978, Sigma) were used to detect protein levels. β-Actin was used as a loading control.