Download Supplementary Information (doc 33K)

Supplementary Materials and Methods
Mass spectrometry
Immunoprecipitates were separated by SDS-PAGE and stained with silver. Specific
silver-stained gel bands were excised, destained, dehydrated, and alkylated. After gel pieces
were dried, proteins were digested with trypsin and peptides were extracted. Each tryptic
peptide sample was dissolved and analyzed by LTQ (Thermo). MS data were searched using
SEQUEST algorithm against the National Center for Biotechnology Information (NCBI)
human protein database and results were filtered, sorted, and displayed using the Bioworks
3.2 softerware.
Construction of recombinant Lentivirus vectors with shRNA for hCINAP
The siRNA sequence for hCINAP (5’-CAGAGUAGUUGAUGAGUUA-3’) was selected after
screening
to
validate
potential
siRNAs
(25).
Non-silencing
siRNA
(5’-UUCUCCGAACGUGUCACGU-3’) was used as a control. Corresponding to siRNA
sequences for hCINAP and non-silencing, shRNAs were synthesized and inserted into
downstream of the U6 promoter in the lentiviral vector pGCSIL-Puromycin (GeneChem Co.,
Ltd., Shanghai, China). To further verify that the effects of hCINAP RNAi are specific, we
prepared two constructs bearing two and three-point mutations in the third base of codons
within the 19-bp RNAi sequence (5’-CAGA180GUA183GTT186GATGAGTTA-3’) targeting
hCINAP expression. The silent mutations in the third base of codon didn’t change the amino
acid sequence of hCINAP protein but rendered its expression insensitive to inhibition by the
shRNA. The RNAi-resistant mutants were named as hCINAP mutant 1 (A180G, A183C),
hCINAP mutant 2 (A180G, A183C, T186C).
Ribosome profiling
At 48 h after transfection with indicated plasmids, HeLa cells were treated with 100 μg/ml
Cycloheximide (CHX) for 10 min. Cells were then washed with pre-cold PBS (containing
100 μg/ml CHX) and resuspended in hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2,
10 mM KCl, 0.5 mM DTT, 100 μg/ml CHX, 40 U/ml RNase Inhibitor, 1 x Cock tail) on ice for
15 min. The cells were mechanically disrupted by 15 strokes with Dounce homogenizer
(KONTES). The homogenate was centrifuged at 720 g for 10 min, and the supernatant was
collected and considered as the cytoplasmic fraction. 1 mg of total proteins was loaded onto a
linear sucrose gradient (5-50%) prepared with a Gradient Master former (BioComp
Instruments). The samples were centrifuged for 150 min at 36,000 rpm at 4°C in a SW41
rotor (Beckman). The gradient was then analyzed at OD254 nm with BioComp Gradient
Fractionator to get ribosome profiles.