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Blood, Vol. 111, Issue 6, 3163-3172, March 15, 2008
Ezrin is a target for oncogenic Kit mutants in murine erythroleukemia
Blood Monni et al.
111: 3163
Supplemental materials for: Monni et al
HS1 or HS2 cells (107 cells) were washed with PBS and lysed in 0.5 ml solubilization buffer (0.3% (w/v) SDS, 50 mM, Tris/HCl,pH7.5, 1mM NaF, 1mM sodium vanadate, 2mM EGTA, 1mM sodium pyrophosphate, 40mM dithiothreitol supplemented with protease inhibitors). Cell extracts were incubated for 30 min on ice with 2.5µg DNAse and 0.5µg RNAse and centrifuged for 30 min at 20 000g. Proteins from the supernatant were precipitated using acetone and dissolved in 450 µl isofocalisation buffer (5M urea, 2M thiourea, 4% (w/v) CHAPS, 50 mM dithiothreitol, 0.5% (v/v) ampholines at pH 4–7 (Amersham Biosciences). Proteins were separated through 24 cm immobilized pH gradient strips (pH 4–7 linear, Amersham Biosciences) for a total of 67 000V×H. After equilibration in SDS-containing buffer, proteins were separated for second dimension on homogenous 11% acrylamide gels. Gels were stained by ammoniacal silver nitrate, digitalized (see figure) and analyzed using the Melanie 4 software. Around 2000 spots were detected in each gel. Among them, spot 56 was overexpressed in HS2 extracts. It was excised and digested with trypsin. Peptides were extracted, cleaned using Zip-tips and analyzed by mass spectrometry using a Voyager DE-PRO MALDI-TOF mass spectrometer. Protein identification was performed by mass finger printing by searching the SwissProt data base using the MASCOT software.
Files in this Data Supplement:
- Table S1. Search parameters and results of the identification (PDF, 12.3 KB)
- Table S2. Phenotypic data from different 663HS1 and 606HS2 or 931HS2 cell clones transfected with expression vectors for either pEF-neo EzWT-VSVG or pEF-neo EzNter-VSVG, pEF-neo EzY145F-VSVG, pEF-neo EzY353F-VSV -
Living and dead cells number: Cells were plated at 2 × 105 cells/mL and cultured in alpha minimun essential medium ( MEM, GibcoBRL) supplemented with 10% fetal calf serum (FCS, GibcoBRL) during 48H. Dead cells were scored by Trypan blue exclusion staining. The living cells numbers and the percentage of dead cells were determined at 48H of culture. The mean number of cells and standard deviations were determined from 3 experiments.
Clonogenicity: Clonogenic assays were performed in 1% methylcellulose medium (MethoCult M3134, Stem cell Technologies, Tebu, France) supplemented with 1% FBS and Epo (1U/mL). Four-hundred growing cells were seeded into 1.5mL medium in 30-mm dishes and colonies (50 cells at least) were counted on day 6 of incubation. Data represent mean ± SD of 6 independent experiments performed in duplicate.
Number of apoptotic cells by Hoechst staining: Cells were fixed with PBS containing 4% paraformaldehyde for 10 min at room temperature, washed in PBS, and stained for 10 min with 50mg/mL Hoechst 33342 (Molecular Probes, Eugene, OR) in PBS. Apoptotic cells were examined under an inverted fluorescence microscope. Fragmented and condensed nuclei in apoptotic cells were scored on three different fields (500 cells per field). Percentage of apoptosis is indicated as mean ± SD of 3 independent experiments.
Tumorigenicity: Cells (1 × 107 in 0.5mL -MEM medium containing 2% FBS) were injected subcutaneously into 8-week-old female nude mice. Tumor nodules were taken off after 3 weeks and weighted.
- Figure S1. 2D gel electrophoresis of HS1 and HS2 cell extracts (JPG, 65.6 KB)
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