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. 2020 Mar 5;16(3):e1008561. doi: 10.1371/journal.pgen.1008561

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© 2020 Zhao et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 5 — (A) FLS2 is a cell body protein and transported to cilia during ciliary disassembly as examined by immunostaining. fls2 cells expressing FLS2-HA were treated with or without NaPPi for 10 min followed by immunostaining with HA and α-tubulin antibodies. WT cells were used as control. Bar, 5 μm. (B) Analysis of ciliary transport of FLS2 by immunoblotting. fls2 cells expressing FLS2-HA were separated into cell bodies (CB) and cilia (C) fractions after treatment with or without NaPPi for 10 min followed by immunoblotting. WC, whole cells. (C) Ciliary FLS2 is associated with the axonemes. Cilia isolated from cells treated with or without NaPPi for 10 min were fractionated into membrane matrix (M+M) and axonemal fractions followed by immunoblotting with the indicated antibodies. FMG1, a ciliary membrane protein was used as a control for M+M fractions. (D) FLS2 is transported to cilia during ciliary disassembly in zygote development. Immunostaining analysis of FLS2 in mt+ and mt- gametes (G+ and G-), 0.5 hr (Z0.5h) and 2.5 hr (Z2.5h) zygotes. Bar, 5 μm. (E) FLS2 in cilia undergoes dephosphorylation during ciliary disassembly but its levels are unchanged. Cilia were isolated from cells treated with NaPPi for 10 or 120 min. The isolated cilia were treated with or without phosphatase (Ptase) followed by phos-tag immunoblotting. Please note that FLS2 in the 10 min sample without phosphatase treatment exhibited slower gel motility shift relative to other samples. (F) The kinase activity of FLS2 is not required for its ciliary transport. Whole cells (WC) or isolated cilia from kinase-dead K33R mutant cells treated with or without NaPPi for 10 min were subjected to immunoblot analysis. (G) FLS1 does not affect ciliary transport of FLS2. Cilia isolated from fls1 cells expressing FLS2-HA treated with or without NaPPi were analyzed by phos-tag immunoblotting. fls2 cells expressing FLS2-HA were used as control. Ciliary transport as well as gel mobility of FLS2 expressed in fls1 were similar to the control.