Fuji imager

Strains used were derived from W303-1a, with P_MET3::RAT1 integrated at the RAT1 locus and carrying a URA3 plasmid (pRS316) to allow growth in medium lacking uracil. Plasmids used are listed in Table 1. The effects of Rat1 depletion were analyzed in this strain additionally transformed with pRS315 (empty plasmid; strain YEAH212), pRS315-RAT1-HA (expressing functional HA-tagged Rat1; strain YEAH213) or pRS315-rat1(D235A)-HA (expressing catalytically inactive, HA tagged Rat1_D235A; strain YEAH214).
Following addition of methionine for 8 h, the Rat1-depleted and complemented strains were pulse-labeled with [³H-5,6] uracil. Cells were harvested at 30 sec intervals by transfer of 900 µl culture samples into 900 µl ethanol at −80°C, to rapidly inhibit label uptake and RNA metabolism. RNA was extracted, separated on denaturing agarose/glyoxal gels and transferred to nylon membranes (Hybond N+). RNA labeled with [³H] uracil was visualized using a Fuji imager (Figure S1). To allow different data sets to be directly compared, signals were normalized to the average values for the 27SA pre-rRNA plateau, which was previously shown to give the most reliable results [7] (link).

Mice were anesthetized with ketamine and xylazine and 4 μl [¹⁴C]C16-ceramide (ARC0831, 55 mCi/mmol) injected into the lumen of the trachea. Prior to injection, [¹⁴C]C16-ceramide was dried, resuspended in 0.5% OGP in 150 mM sodium acetate (pH 7.4 or 5.0), and bath sonicated for 10 min. After 20–30 min, mice were sacrificed, the trachea was carefully removed and extracted in 200 μl H₂O and CHCl₃:CH₃OH:HCl (100:100:1, v/v/v). The lower phase was dried, samples were resuspended in CHCl₃:CH₃OH (1:1, v/v) and separated by TLC using CHCl₃:CH₃OH:ammoniumhydroxide (90:20:0.5, v/v/v) as the developing solvent. The TLC plates were analyzed using a Fuji-Imager.

OSM‐3 was desalted using a Micro Bio‐spin 6 column (BioRad, Marnes‐La‐Coquette, France) to remove excess nucleotide. OSM‐3 (5 µm) was then incubated with 15 µm pig brain tubulin at 37 °C during 30 min in a microtubule‐assembly buffer (50 mm Mes‐K, pH 6.8, 30% glycerol, 0.5 mm EGTA, 6 mm MgCl₂, 0.5 mm GTP), in the absence or in the presence of 2 mm ADP or 1 mm AMPPNP. After high speed centrifugation (300 000 g for 15 min at 35 °C), the supernatant and pellet were analyzed by SDS/PAGE stained with Coomassie blue.
In the case of the 1–337 construct, the identity of the kinesin band was confirmed by western blot (Fig. 1D). After SDS/PAGE separation, the proteins were transferred to a nitrocellulose membrane. The membrane was then blocked in 5% skimmed milk and incubated with a 1 : 2000 dilution of an anti‐His antibody (Sigma A7058, St. Quentin Fallavier, France). The blot was developed with a chemiluminescent substrate (Super Signal West Pico; Thermo, Les Ulis, France) and the proteins were visualized using a Fuji imager.

Cells were lysed in 1% Nonidet P40 (NP40) in 150 mM sodium acetate (pH 4.5) for 5 min on ice, diluted to 0.1% NP40 in 150 mM sodium acetate (pH 4.5) and 0.3 μCi/sample [¹⁴C₁₆]ceramide (ARC0831, 55 mCi/mmol) was added. To this end, the [¹⁴C₁₆]ceramide was dried for 10 min, resuspended in 0.1% OGP in 150 mM sodium acetate (pH 4.5) and bath sonicated for 10 min. The samples were incubated at 37°C for 60 min. The reaction was terminated by extraction in H₂O and CHCl₃:CH₃OH:HCl (100:100:1, v/v/v). The lower phase was dried, and samples were resuspended in CHCl₃:CH₃OH (1:1, v/v), separated by TLC with CHCl₃:CH₃OH:NH₄OH (90:20:0.5, v/v/v), and analyzed with a Fuji Imager.

HEK293T cells were cotransfected with HIV-1-NL4.3 and increasing amounts of IFITM plasmids. pQCXIP-empty was used as a control and to ensure all transfections had an equivalent amount of total plasmid during transfection. At 48 hours post-transfection, HEK293T cells were harvested in RIPA buffer (containing 0.05% SDS). Cell debris was removed by centrifugation. A fraction of the lysate was used for total input analysis by Western blot while the rest was subjected to immunoprecipitation using anti-FLAG beads. Immunoprecipitations were incubated overnight at 4˚C with rotation. Beads were centrifuged and washed three times with cold lysis buffer. All samples were boiled in sample buffer prior to resolving by SDS-PAGE. The gels were transferred to polyvinylidene difluoride (PVDF) membranes and blocked in blocking solution (5% non-fat milk in PBS 0.1% Tween-20) for 1 hour at room temperature. Membranes were washed (PBS with 0.1% Tween-20) three times followed by overnight incubation with the primary antibody in blocking solution overnight at 4˚C. The membranes were washed three times and incubated with secondary antibodies conjugated to HRP for 1 hour at room temperature. The blots were incubated with HRP substrate (Millipore # WBLUR0100) and bands were visualized using a Fuji Imager.