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1 year ago

Stattic DNA Synthesis inhibitor Paclitaxel

The
two bright green fluorescence dots on the proximal base of the flagella are KMP11 signal inside the Stattic DNA Synthesis inhibitor Paclitaxel basal
bodies. Scale bar: 5 ��m. (B) Stability of CC2D and KMP11 in FAZ2 RNAi cells. FAZ2 RNAi cell line expressing endogenously PTP-tagged KMP11 was induced with tetracycline for 72 h or with tetracycline
for 72 h and MG-132 for 8 h. Soluble and pellet fractions have been immunoblotted with anti-CC2D and anti-
Protein A to detect CC2D and KMP::PTP, respectively. The same membrane was re-blotted with anti-��-
tubulin and anti-TbPSA6. Protein band intensity was determined as described in Fig. 2, and plotted being a
histogram shown beneath the Western blots. Error bars signify S.D. calculated from 3 independent
experiments. S: cytosolic fraction; P: cytoskeleton fraction.

(C) Result of KMP11 RNAi on FAZ2 and
CC2D localization. FAZ2 was endogenously tagged using a triple HA epitope in KMP11 RNAi cell line.
Cells were co-immunostained Stattic DNA Synthesis inhibitor Paclitaxel with FITC-conjugated anti-HA mAb and anti-CC2D pAb. White
arrowheads display the FAZ2::3HA and CC2D in the proximal base from the detached new flagellum (black
arrow). Scale bar: 5 ��m. (D). Stability of FAZ2 and CC2D in KMP11 RNAi cells. KMP11 RNAi cell line expressing FAZ2::3HA was induced with tetracycline for 32 h or with tetracycline for 32 h and MG-132
for 8 h. Soluble and pellet fractions were immunoblotted with anti-CC2D pAb and anti-HA mAb to detect
CC2D and FAZ2::3HA, respectively. The identical membrane was re-blotted with anti-��-tubulin and anti-
TbPSA6 as cytoskeleton and cytosol markers, respectively.

Protein band intensity was established as
described in Fig. 2, and plotted like a histogram proven under the Western blots. Error bars indicate S.D.
calculated from three independent experiments. S: cytosolic fraction; P: cytoskeleton fraction.
Fig. 6. CC2D depletion destabilizes FAZ2 and KMP11. (A). Effect of CC2D RNAi over the localization
of FAZ2. FAZ2 was endogenously tagged using a C-terminal triple HA epitope in cells harboring a CC2D
RNAi construct. Cells had been immunostained with FITC-conjugated anti-HA mAb. The white arrow demonstrates
the FAZ2::3HA in the proximal base of detached new flagellum, along with the black arrow signifies the
detached new flagellum. Scale bar: 5 ��m. (B) Effect of CC2D RNAi on KMP11 localization. KMP11 was
endogenously tagged with an Stattic DNA Synthesis inhibitor Paclitaxel HA tag in CC2D RNAi cell line.

Cells had been co-immunostained with FITC-conjugated anti-HA mAb and anti-CC2D pAb. White arrows indicate the KMP11::HA from the FAZ
filament, whereas the white arrowhead exhibits the CC2D signal at the proximal base with the detached new
flagellum (black arrow). Scale bar: 5 ��m. (C, D). Stability of FAZ2 (C) and KMP11 (D) in CC2D RNAi
cells. CC2D RNAi cell line expressing endogenously 3HA-tagged FAZ2 or PTP-tagged KMP11 was
induced with tetracycline for 72 h or with tetracycline for 72 h and MG-132 for 8 h.

1 year ago

Stattic DNA Synthesis inhibitor Paclitaxel

(A). Two-dimensional DiGE
examination of FAZ2 RNAi non-induced and induced cytoskeletons. Proven are the 2D pictures of 3
representative proteins, FAZ8, Tb927.11.2610, in non-induced and FAZ2 RNAi induced cells. The 2D
gel was analyzed making use of DeCyder application to make three-dimensional images in the three protein spots
that Stattic DNA Synthesis inhibitor Paclitaxel demonstrate a reduction in volume soon after FAZ2 RNAi induction. (B-D). Amounts of FAZ8, Tb927.eleven.2610,
and PFC7 in FAZ2 RNAi cells. FAZ8 (B), Tb927.11.2610 (C), and PFC7 (D) were each and every endogenously
tagged with a triple HA epitope in FAZ2 RNAi cells. The three proteins have been detected with anti-HA
antibody. ��-tubulin and TbPSA6 were integrated as cytoskeleton and cytosol markers, respectively. Protein
band intensity was determined as described in Fig.

2, and plotted as histograms proven beneath the Western
blots. Error bars indicate S.D. calculated from three independent experiments. S: cytosolic fraction; P: cytoskeleton fraction. (E). Immunostaining of 3HA-tagged FAZ8 in manage and FAZ2 RNAi cells.
White arrows indicate the quick, new FAZ filament (snFAZ) linked with all the detached new flagellum
(black arrows). Scale bars: 5 ��m. (F). Stability of 3HA-tagged FAZ8 in non-induced management and FAZ2
RNAi cells. FAZ2 RNAi was induced with tetracycline for 72 h or with tetracycline for 72 h and MG-132
for 8 h. Immunoblotting was carried out with anti-HA mAb. Exactly the same blot was re-probed with anti-��-
tubulin and anti-TbPSA6. Protein Stattic DNA Synthesis inhibitor Paclitaxel band intensity was established as described in Fig. 2, and plotted being a
histogram proven under the Western blots.

Error bars represent S.D. calculated from three independent
experiments. S: cytosolic fraction; P: cytoskeleton fraction. Fig. 4. FAZ2 varieties a complicated with CC2D and KMP11. (A, B) Co-immunoprecipitation of CC2D by
FAZ2 (A) and KMP11 (B). FAZ2::PTP and KMP11::PTP had been precipitated by incubating with IgG
sepharose beads, as well as immunoprecipitate was immunoblotted with anti-CC2D and anti-Protein A (��-
ProtA) to detect CC2D and PTP-tagged FAZ2 and KMP11, respectively. PTP-tagged ��-tubulin (��-
TUB::PTP) was included as a detrimental handle. (C). Co-immunoprecipitation of FAZ2::3HA by
KMP11::PTP. FAZ8::3HA and KMP11::PTP have been co-expressed from their respective endogenous locus
from the very same cell line. Cells expressing FAZ2::3HA or KMP11::PTP served as controls. The asterisk
showed a non-specific band Stattic DNA Synthesis inhibitor Paclitaxel detected by anti-HA antibody from the input, but not within the immunoprecipitates.
Fig. 5. FAZ2 RNAi destabilizes CC2D and KMP11, and KMP11 RNAi destabilizes FAZ2 and
CC2D. (A) FAZ2 depletion on CC2D and KMP11 localization. KMP11 was endogenously tagged in
FAZ2 RNAi cells. Cells had been co-immunostained with FITC-conjugated anti-HA mAb and anti-CC2D
pAb.

1 year ago

Stattic DNA Synthesis inhibitor Paclitaxel

PLoS 1 5, e9660.
Zhou, Q. and Li, Z. (2014). Polarized assembly and assembly-associated Stattic DNA Synthesis inhibitor Paclitaxel protein stability on the
falgellum attachment zone filament in Trypanosoma brucei. The 25th
Molecular Parasitology Meeting,
Conference proceedings, Woods Hole, MA, pp268.
Zhou, Q., Liu, B., Sun, Y. and He, C. Y. (2011). A coiled-coil- and C2-domain-containing protein is
required for FAZ assembly and cell morphology in Trypanosoma brucei. J Cell Sci 124, 3848-58. Fig. 1. FAZ2 is a new FAZ protein needed for flagellum adhesion. (A) FAZ2 localizes to your FAZ
filament. Cells expressing FAZ2::3HA were co-immunostained with FITC-conjugated anti-HA mAb and
anti-FAZ1 mAb or anti-CC2D pAb. Scale bars: 5 ��m. (B) Depletion of FAZ2 by RNAi.

FAZ2 was
endogenously tagged with a triple HA epitope in cells harboring the FAZ2 RNAi construct. Cells had been handled with PEME plus 1% NP40, and cytosolic (S) and cytoskeletal (P) fractions had been separated and
detected by immunoblotting Stattic DNA Synthesis inhibitor Paclitaxel with anti-HA antibody. Exactly the same blot was re-probed with ��-tubulin and
TbPSA6 as cytoskeleton and cytosol markers, respectively. (C) FAZ2 knockdown inhibits cell
proliferation. (D) Quantification of cells with distinctive numbers of nucleus (N) and kinetoplast (K) upon
FAZ2 RNAi. Error bars represent S.D. calculated from 3 independent experiments. (E) Percentage of
cells with detached flagella upon FAZ2 RNAi. Error bars represent S.D. from 3 independent
experiments. Fig. 2. FAZ2 depletion destabilizes FAZ proteins. (A, D). Immunostaining of FAZ1 and
Tb927.7.

3330::3HA in manage and FAZ2 RNAi cells. Tb927.73330 was tagged at its endogenous locus
using a C-terminal triple HA epitope in FAZ2 RNAi cells. White arrows indicate the quick, new FAZ
filament (snFAZ) connected using the detached new flagellum (black arrows). Scale bars: 5 ��m. (B, E).
Ranges of FAZ1 (B) and Tb927.7.3330 (E) in FAZ2 RNAi cells. The two genes were each and every endogenously
tagged that has a triple HA epitope in FAZ2 RNAi cells, and detected by anti-HA mAb. The exact same blot was
probed with ��-tubulin and TbPSA6 as cytoskeleton and cytosol markers, respectively. Protein band
intensity was established, normalized with that of loading controls, and plotted as histograms proven
beneath the western blots. Error bars signify S.D. calculated from three independent experiments.

S:
cytosolic fraction; P: cytoskeleton fraction. (C, F). Stability of 3HA-tagged FAZ1 and Tb927.7.3330 in
FAZ2 RNAi cells. FAZ2 RNAi was Stattic DNA Synthesis inhibitor Paclitaxel induced with tetracycline for 72 h or with tetracycline for 72 h and MG-132 for 8 h. FAZ1 and Tb927.7.3330 have been detected by anti-HA mAb. Exactly the same blot was re-probed
with anti-��-tubulin and anti-TbPSA6, respectively. Protein band intensity was established as described
above. Error bars indicate S.D. calculated from 3 independent experiments. S: cytosolic fraction; P:
cytoskeleton fraction. Fig. 3.

1 year ago

Stattic DNA Synthesis inhibitor Paclitaxel

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Bonhivers, M., Nowacki, S., Landrein, N. and Robinson, D. R. (2008). Biogenesis in the
trypanosome endo-exocytotic DNA Synthesis signaling organelle is cytoskeleton mediated. PLoS Biol 6, e105.
Broadhead, R., Dawe, H. R., Farr, H., Griffiths, S., Hart, S. R., Portman, N., Shaw, M. K., Ginger,
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