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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Mol Microbiol. 2015 Sep 4;98(4):667–680. doi: 10.1111/mmi.13149

Figure 6. GCP2 RNAi disrupts Hydin and PF16 assembly to the new flagellum.

Figure 6

(A, B). Effect of GCP2 RNAi on the localization of Hydin (A) and PF16 (B). Hydin and PF16 were each endogenously tagged with a triple HA epitope in cells harboring the GCP2 RNAi construct. Non-induced control and GCP2 RNAi-induced cells (36 h and 72 h) were immunostained with anti-HA antibody to detect Hydin::3HA and PF16::3HA. The white bracket shows the reduced Hydin::3HA signal in the middle portion of the new flagellum, the white arrows show the portions of the new flagellum with reduced PF16::3HA signal, and the white arrowheads indicate the signal of Hydin::3HA and PF16::3HA in the detached flagellum (black arrows). Scale bars: 5 μm. (C). GCP2 RNAi caused random depletion of PF16::3HA from the new flagellum at earlier time points of RNAi induction. White arrows show the PF16::3HA signal (as multiple dots) at the distal portion of the new flagellum, and white brackets outline the portions of the new flagellum with much reduced PF16::3HA signal. Scale bar: 5 μm. (D, E). Effect of GCP2 RNAi on the stability of Hydin (D) and PF16 (E) in cytosolic and the cytoskeletal fractions. Non-induced control and GCP2 RNAi cells treated without and with MG-132 were lysed in PEME buffer containing 1% NP-40 for cytoskeleton preparation. The soluble cytosolic fraction (S) and cytoskeletal pellet fraction (P) were separated by centrifugation, loaded onto a SDS–PAGE gel and transferred onto a PVDF membrane for western blotting with anti-HA antibody to detect Hydin::3HA and PF16::3HA. The same blot was probed with anti-TbPSA6 as the cytosolic marker and with anti-α-tubulin as the cytoskeletal marker. Similar results were obtained in three independent experiments.