(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