Easytag

Cells were incubated for 30 min in DMEM without methionine and cysteine supplemented with 1% dialysed FCS, pulsed with ³⁵S-labeled amino acids (200 mCi/9*10⁶ cells) (Easy Tag, Perkin Elmer), washed and chased in complete medium for the indicated times. After different times, cells were treated with 10 mM NEM and lysed in RIPA as described [48] (link). Immunoprecipitates were resolved on SDS–PAGE under reducing conditions, transferred to nitrocellulose and membranes visualized by autoradiography with FLA900 Starion (FujiFilm Life Science, Tokyo, Japan). Subsequently, the red signal form TMR was visualized with infrared technology. Densitometric quantifications of the signals were performed with ImageJ.

Full-length myc-KIF1C synthesized in vitro with ³⁵S-methionine (EasyTag, Perkin Elmer, San Jose, CA) was desalted and incubated with GTPγS-preloaded Rabs (4.2 μM) for 1 hr in Buffer A (−BSA), 1 mM DTT, 2.5 mM ADP, 0.5 mM GTPγS. Microtubules (0.8 mg/ml), polymerized in 80 mM PIPES, 1 mM MgCl₂, 1 mM EGTA, pH 6.8 (BRB80), 1 mM DTT, 1 mM GTP, 10% DMSO, spun through a 40% glycerol cushion, and resuspended in BRB80, 1 mM DTT, 0.2 mM Paclitaxel (Cytoskeleton, Inc.), were incubated with the KIF1C-Rab complexes for 1 hr before being spun through a 10% sucrose, 20 µM Paclitaxel, 1 mM DTT, at 65K for 5 min (Optima TLX, Beckman Coulter, Inc., Indianapolis, IN). Scintillation counting and SDS-PAGE and radiography using a Typhoon imager (GE Healthcare Biosciences) revealed the amount of ³⁵S-labeled-KIF1C in fractions.

Mitochondrial protein synthesis in cultured cells was detected through metabolic labeling with [³⁵S] methionine/cysteine in the presence of anisomycin to inhibit cytoplasmic ribosomes [38 (link)]. After treatment with targeted or control siRNA for 5 days, cells were washed three times with PBS and pretreated with 100 µg/mL anisomycin for 5 min to inhibit cytoplasmic translation. Subsequently, [³⁵S] methionine/cysteine (EasyTag, PerkinElmer) was added with a final concentration of 400 μCi for pulse labeling assays. After pulse labeling for 30 min, cells were washed twice either with PBS (pulse samples) or medium without [³⁵S] methionine/cysteine (chase labeling). After 6 h of chasing, cells were scraped and treated with benzonase® Nuclease (E1014, Sigma) according to the manufacturer’s instructions. Gel loading buffer (186 mM Tris–HCl, pH 6.7, 15% glycerol, 2% SDS, 0.5 mg/mL bromophenol blue, and 6% -mercaptoethanol) was added to the samples before loading onto a 12–20% gradient SDS-PAGE gel to separate proteins. The gel was then dried for exposure to a phosphor screen and scanned with a FUJIFILM FLA-5100. Gels were rehydrated in water and Coomassie-stained to confirm equal loading.

Following the indicated treatments, 293T cells were incubated for 20 minutes in 90% methionine/cysteine-free DMEM (ThermoFisher Scientific) complemented with 5% dialyzed serum and 2 mM L-glutamine. Cells were then incubated for 1 hour in the same medium in the presence of 100 μg/ml emetine (Sigma-Aldrich) to inhibit cytoplasmic translation and 100 μCi/ml ³⁵S-labeled methionine/cysteine (EasyTag, PerkinElmer). Lysates were resolved on a 12% Bolt Bis-Tris Plus gel (ThermoFisher Scientific) and analyzed by autoradiography.