cell line: HeLa BAC KIF1C-GFP #3843 antibody: GFP-Trap
Treatment protocol
Cells were rinsed in ice-cold PBS, and all subsequent manipulations were performed at 4°C. Cells were scraped in HNTG buffer (20 mM HEPES-KOH pH 7.9, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM MgCl2, 1 mM EGTA), containing an antiprotease cocktail (Roche). Cells were incubated for 20 minutes on a rotating wheel, and cellular debris were the removed by centrifugating the extracts 10 minutes at 20,000g. Beads coated with GFP-trap antibody (ChromoTek), or uncoated as control, were washed in HNTG (25 l of beads per IP). Beads were incubated 1h with a control extract to saturate non-specific binding and then incubated with the proper extract. After 4h of incubation on a rotating wheel, beads were washed four times in HNGT with anti-protease, and twice with PBS.
Growth protocol
HeLa cells stably expressing the a KIF1C-GFP fusion from a Bacterial Artifical Chromosome (BAC) were grown to near confluence in 10 cm plates in DMEM, 10% FCS, at 37°C, and two plates were used per Immunoprecipitation (IP),
Extracted molecule
total RNA
Extraction protocol
Beads were then incubated with Trizol to extract RNAs, and RNA purification was done as recommended by the manufacturer.
Label
biotin
Label protocol
The resulting RNAs were amplified and converted into cDNAs by the WT PICO kit (Thermo Fisher).
Hybridization protocol
Samples were hybridized with GeneChip Human Transcriptome Array 2.0 chips (Affymetrix) on an Affymetrix platform (Thermo Fisher).
Scan protocol
Array scanning was performed on a GeneChip Scanner 3000 7G according to the manufacturer's instruction (Affymetrix).
Data processing
Affymetrix Expression Console software version 1.4.1.46 was used ProcessedDatas_KIF1C.xls contains the processed signal intensity for IP-KIF1C over IP-Control for each affymetrix probesets.
