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Supplementary Fig. 1.
certain cell lines.
Absence of 45 kD protein recognized by HD-2 antiserum in
(a) Loss of 45 kD protein expression in the RBT and RBR
tumorigenic cell lines determined by Western blot analysis. (b) Expression of 45 kD
protein in various cancer cell lines analyzed by Western blot method.
MeWo
(melanoma cell line) produced high level of 45 kD protein, but AZ521 (stomach cancer
cell line) did not.
Other human cancer cell lines, such as HT1080 (fibrosarcoma),
G401 (Wilm’s tumor), MIA-PaCa (pancreatic cancer), CaSki (cervical cancer), C33A
(cervical cancer), T24 (bladder cancer), were also analyzed.
Supplementary Fig. 2.
Identification of 45 kD protein as CapG.
(a) Two-dimensional
gel electrophoresis of total cell lysates prepared from AZ521 and MeWo. Upper panel,
coomasie brilliant blue (CBB) staining; lower panel, Western blot (WB). (b) Amino
acid sequence of CapG protein and nine tryptic peptides (underlined) determined by
mass spectrometric analysis.
Supplementary Fig. 3.
antibody.
Recognition of CapG protein by a newly prepared anti-CapG
(a) Detection of GST-CapG, but not GST, by the anti-CapG antibody.
Left
panel, CBB staining; right panel, WB. (b) Detection of endogenous CapG protein by
the antibody.
U937, human histiocytic lymphoma cell line, has been reported to
differentiate into macrophage-like cells by treatment with PMA (100nM) for 2 days and
to express high level of CapG protein (Dabiri et al. 1992).
Supplementary Fig. 4.
Ubiquitous expression of CapG mRNA in normal human
tissues. Human RNA blot (Clontech) was subjected to Northern blot hybridization
with 32P-labeled CapG cDNA. RNA blotted were obtained from 1A: whole brain; 1B:
cerebral cortex; 1C: frontal lobe; 1D: parietal lobe; 1E: occipital lobe; 1F: temporal
lobe; 1G: paracentral gyrus of cerebral cortex; 1H: pons; 2A: left cerebellum; 2B: right
cerebellum; 2C: corpus callosum; 2D: amygdala; 2E: caudate nucleus; 2F:
hippocampus; 2G: medulla oblongata; 2H: putamen; 3A: substantia nigra; 3B:
accumbens nucleus; 3C: thalamus; 3D: pituitary gland; 3E: spinal cord; 4A: heart; 4B:
aorta; 4C: left atrium; 4D: right atrium; 4E: left ventricle; 4F: right ventricle; 4G:
interventricular septum; 4H: apex of the heart; 5A: esophagus; 5B: stomach; 5C:
duodenum; 5D: jejunum; 5E: ileum; 5F: ilocecum; 5G: appendix; 5H: ascending colon;
6A: traverse colon; 6B: descending colon; 6C: rectum; 7A: kidney; 7B: skeletal muscle;
7C: spleen; 7D: thymus; 7E: peripheral blood lymphocyte; 7F: lymph node; 7G: bone
marrow; 7H: trachea; 8A: lung; 8B: placenta; 8C: bladder; 8D: uterus; 8E: prostate; 8F:
testis; 8G: ovary; 9A: liver; 9B: pancreas; 9C: adrenal gland; 9D: thyroid gland; 9E:
salivary gland; and 9F: mammary gland.
Ubiquitin expressed in all tissues was used
as an internal control.
Supplementary Fig. 5.
Subcellular localization of CapG protein in the RB and AZ521
cell lines. The cell lines indicated were fixed with 2% paraformaldehyde, incubated
with anti-CapG antibody, followed by incubation with Alexa 488-labeled anti-rabbit
IgG (Molecular Probes, Eugene, USA), and then stained with DAPI.