Odyssey software

Parasite lysates were prepared from P. knowlesi schizont as described previously [55 (link)]. Recombinant proteins were separated by 13% SDS-PAGE under reducing or non-reducing conditions and stained with 0.25% Coomassie brilliant blue R-250 (Sigma-Aldrich) and used for western-blotting analysis [25 (link)]. The membrane-transferred proteins were reacted with primary rabbit polyclonal serum (1:50) or an anti-His monoclonal antibody (1:2,000, Hilden, Hamburg, Germany) and then reacted with secondary IRDye-labeled goat anti-rabbit or goat anti-mouse antibodies (1:10,000) (LI-COR Bioscience, Lincoln, NE). An Odyssey infrared imaging system (LI-COR Bioscience) and Odyssey software (LI-COR Bioscience) were used to visualize the bands.

To examine 8-oxoG lesions after H₂O₂ treatment, U2OS cells were lysed in buffer (100 mM Tris, pH 7.6, 200 mM NaCl, 0.5 mM EDTA, pH 8.0, 0.5% SDS (w/v), 0.5% Triton X-100 (v/v), 0.2 mg/mL Proteinase K, 0.1 mM Deferoxamine mesylate salt (Sigma)) containing RNase A (Roche) and incubated at 55°C overnight. Chromosomal DNA was precipitated with isopropanol and re-suspended in TE buffer. DNA concentration was determined using a Nanodrop 2000 (Thermofisher). Equal amounts of DNA were immobilized on a nylon membrane using a 48-well Slot blot vacuum manifold. DNA was cross-linked onto the membrane using a UV Stratalinker before blocking with 2% v/v fish gelatin in PBS-T (0.05% Tween 20). Membranes were probed with the 8-oxoG antibody, washed with PBS-T and probed with an appropriate secondary antibody. Membranes were scanned on an Odyssey infrared imaging system (LiCor). Signal intensity was assessed by densitometry using the Odyssey software (LiCor). Results are displayed graphically as mean ± S.D. and significance was examined using a Student's t test with a P value of <0.05 considered significant.

Total RNA from the cell lines and primary cultures was isolated with TRIzol reagent (Invitrogen, Barcelona, Spain). cDNA synthesis was performed as described elsewhere [21 (link)]. Pit-1, BRCA1, RAD1, RAD18, RAD51, RAD52, RAD54B, GADD45A, GADD45B, GADD45G, vitamin D receptor (VDR) and 18S mRNA levels were quantified using real-time PCR. Microarray assay of mRNA was performed using an Affymetrix Human Gene 1.0 ST Array (GEO database access no. GSE64101). Western blotting was performed as previously described [21 (link)]. Relative protein expression was quantified using ImageJ software (National Institutes of Health, Bethesda, USA). Pit-1 quantification in primary cultures of human breast tumors was performed using the LI-COR Odyssey software (Homburg, Germany). Primers and antibodies are described in Supporting Information.

Five microgram of protein from either isolated synaptoneurosomes or crude homogenate was loaded onto NuPAGE 4–12% Bis‐Tris precast polyacrylamide 15 well gels (Invitrogen, Paisley, UK) along with molecular weight marker (Li‐Cor, Cambridge, UK). Proteins were electro‐transferred to nitrocellulose membrane (Bio‐Rad, Hemel Hempstead, UK). Membranes probed with the following primary antibodies: Aβ(82E1,IBL,1 : 100), Tau13 (MMS‐520R‐500, Covance, 1 : 2000), β‐actin (ab8226, Abcam, 1 : 2000), Synaptophysin (AB8049, Abcam, 1 : 10 000), α‐tubulin (ab4074, Abcam, 1 : 1000), GFAP (0334, DakoCytomation, 1 : 500), GAPDH (ab8245, Abcam, 1 : 2000). Proteins were visualized on an odyssey infrared system using the appropriate 680 and 800 IR dye secondary antibodies (1 : 50 000, LI‐COR Biosciences) and were analyzed using odyssey software (LI‐COR Biosciences).

siRNA-transfected cells were lysed in RIPA buffer (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 1% Triton X-100, and 0.1% SDS). The lysates were kept on ice for 15 minutes and then clarified of debris by centrifugation. The lysates were incubated with SDS-PAGE sample buffer at 100°C for 10 minutes, and proteins were resolved on a 4%–20% SDS-PAGE gradient gel (Bio-Rad Laboratories, Hercules, CA). The samples were transferred onto iBlot nitrocellulose membranes (Life Technologies, Carlsbad, CA) and blocked for 1 h at room temperature with a blocking buffer (LI-COR Biosciences, Lincoln, NE). The blots were incubated with primary antibodies at 4°C overnight and then with anti-rabbit IRDye 800CW and anti-mouse IRDye 680LT (LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature. Protein bands were visualized using Odyssey software (LI-COR Biosciences, Lincoln, NE).