Supplementary Notes Patients and Methods

advertisement
Supplementary Notes
Patients and Methods
Consenting and exome sequencing
A CMT family with eight individuals was ascertained. Informed consent was
obtained from all individuals and the Institutional Review Boards at the
participating medical centers approved the study. Neurological exam and
neurophysiological studies were performed. Genomic DNA from three affected
subjects was used for whole exome sequencing. The SureSelect Human All Exon
50Mb kit Version 5 (Agilent, Santa Clara, CA , USA) was used for in-solution
enrichment of genomic DNA. Enriched DNA was performed on the HiSeq2000
instrument (Illumina, San Diego, CA, USA) to produce 100bp paired-end sequence
reads. BWA2 was used to align sequence reads to the hg19 human reference
sequence. GATK3 was used for local realignment, base recalibration, and calling of
genotypes. VCF files were then imported into GEM.app4 for further analysis. In
addition, parametric linkage analysis was performed using the Merlin software14.
Protein purification
Human p97 plasmid (TCB197) was subjected to site-directed mutagenesis with
primers containing mutations to create R155H (TCB210), A232E (TCB211), and
E185K (TCB374). Proteins were purified as described (ref).
In vitro ATPase Assay
Purified p97 (12.5 L of 50 M; final concentration in the reaction was 25 nM) was
diluted in 20 mL of assay buffer [5 mL of 5x assay buffer A (1x = 50 mM Tris pH 7.4,
20 mM MgCl2, 1 mM EDTA,) mixed with 15 mL water and 25 L 0.5M TCEP, 25 L
10% Triton] to make the enzyme solution. 40 L of enzyme solution was dispensed
into each well of a 96 well plate. The ATPase assay was carried out by adding 10 L
of 1000 M ATP (Roche, pH 7.5) to each well and incubating the reaction at room
temperature for 25 min. Reactions were stopped by adding 50 L of BIOMOL Green
reagent (Enzo Life Sciences). Absorbance at 635 nm was measured after 4 min on
the Synergy Neo Microplate Reader (BioTek).
Plasmids, transfection and cell culture
The cDNA of mouse p97/VCP was PCR amplified and cloned into EGFPN1
expression vector (Clontech, Palo Alto, CA). The generation of GFP-fused p97/VCP
vector is previously described1. IBMPFD mutations i.e. VCP-A232E (VCP-AE) and
VCP-E185K (VCP-EK) were introduced into this vector using site-directed
mutagenesis kit (Stratagene, La Jolla, CA) and sequenced for the verification of their
point mutations. These GFP-fused constructs containing each p97/VCP point
mutations were transfected into U2OS cells using lipofectamine 2000 (Invitrogen,
Carlsbad, CA) according to the manufacturer’s protocol. The cells were maintained
in DMEM (Gibco, Grand Island, NY) with penicillin/streptomycin and 10% fetal
bovine serum (Atlanta Biologicals, Flowery Branch, GA) at 37 °C with 5% CO2. On
the day of transfection, the cells were seeded and grown to ~80% confluent. 48
hours after the transfection, the cells were washed with PBS (Gibco, Grand Island,
NY) three times and harvested for analysis.
Western blots
Transfected and non-transfected U2OS cells were lysed in RIPA buffer with protease
inhibitor cocktail (Sigma-Aldrich, St.Louis, MO). The lysates were centrifuged at 4 °C
x14,000 g for 15 mins. After the centrifugation, the supernatant were transferred to
a new tube and the concentration of proteins was measured using a BCA assay kit
(Thermo Fisher Scientific, Waltham, MA). The samples were resuspended in
3xLamelli buffer and 20 – 50 μg proteins of proteins were separated on 10 – 15%
SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Bio-Rad
Laboratories, Hercules, CA). The membranes were blocked in PBS with 0.1% Tween20 (PBS-T; Sigma-Aldrich) and 5% non-fat dry milk for 1 hour at room temperature.
After three times washing with PBS-T, the membranes were incubated for overnight
at 4 °C with the following antibodies: anti-p97/VCP (Fitzgerald, Acton, MA), anti-p62
(Proteintech, Chicago, IL), anti-actin (Sigma-Aldrich), and anti-LC3 (Sigma-Aldrich).
The membranes were washed three times with PBS-T and then incubated with
horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG secondary
antibodies for 1 hour at room temperature. The bands were visualized by ECL (BioRad Laboratories).
Download