Pbs buffer

Surface plasmon resonance (SPR) was measured using a Proteon XPR36 instrument (Bio-Rad). The measurements were performed at 30°C in a PBS buffer (Bio-Rad) added with 0.01% of Tween 20 (PBST) and with or without ATP 0.5 mM. NLC sensor chips (Bio-Rad) were used to immobilize the oligonucleotides oso13 or oso15 (Supplementary Table S1) through their biotin-tag. For immobilization, DNA were diluted in PBST and attached to the chip to obtain 50 RU in different orientations (3′ free for oso13 or 5′ free for oso15). For the observation of kinetic data, two different kinds of experiments were performed. In experiments where loading and translocation were coupled, proteins were injected in PBST with 0.5 mM ATP and 10 mM MgCl₂ at 50 μl/min during 240 sec, and dissociation was run during 900 sec. In experiments where loading and translocation of the helicase on DNA were uncoupled, proteins were injected in PBST with 0.5 mM ATP during same duration, and the 900 sec dissociation was followed by injection of PBST with ATP 0.5 mM plus MgCl₂ 10 mM during 459 sec. In all experiments, the proteins were injected at different concentrations and different molar ratios, as indicated in the Results section. After each interaction test, the chip was regenerated using 0.5% of SDS. After corrected by subtraction of the uncoated reference channel, the sensorgrams were analyzed and compared.

Treated cells in 6-cm dishes were washed with ice-cold phosphate-buffered saline (PBS) containing sodium orthovanadate 2 mM (to preserve the protein tyrosyl phosphorylation state in cells and cells lysates) [25 (link)], collected, resuspended in lysis buffer, and processed as previously described [15 (link), 16 (link)]. Briefly, lysates were vortexed, incubated at 4 °C for 30 min, and centrifuged at 13,000rpm for 15 min at 4 °C. The supernatant was collected and sample buffer containing DTT 0.1 M was added. Proteins were resolved by SDS-PAGE on a 12% polyacrylamide gel and then electrophoretically transferred to a polyvinyllidene fluoride (PVDF) membrane (Millipore) using a semi-dry transfer device (Trans-Blot, Bio-Rad, Hercules, CA, USA). Membranes were subsequently blocked with casein 1% in PBS buffer (Bio-Rad, Hercules, CA, USA) for 2 h at room temperature, incubated with the appropriate primary antibody at 1:1000 dilution overnight at 4 °C. Membranes were incubated with infrared-labeled secondary antibodies (anti-mouse 680 Alexa, Molecular Probes, Eugene, OR, USA) or anti-rabbit IRDYE 800 (Rockland Immunochemicals, Gilbertsville, PA, USA) at 1:15,000 dilutions for 2 h at room temperature. Specific protein bands were detected and quantified using Odyssey Infrared Imaging System and Software version 1.2 from Li-Cor Biosciences (Lincoln, NE, USA).