Journal: Cell reports

Article Title: Rab11-Rab8 cascade dynamics in primary cilia and membrane tubules.

doi: 10.1016/j.celrep.2024.114955

Figure Lengend Snippet: Figure 1. Rab8 ciliary trafficking is promoted by Rabin8 and Rab11 (A) Immunoblot analysis comparing lysates from RPE WT, tRFP-Rab8a, tRFP-Rab8a+GFP-Rab11a, tRFP-Rab8a+GFP-Rabin8, tRFP-Rab8a+GFP-Ra- bin8+miRFP-Rab11a, tRFP-Rab8a+GFP-Rabin8+miRFP-Rab14 cells ± Dox probed with Rab8a antibody; RPE WT, tRFP-Rab8a+GFP-Rabin8, tRFP-Rab8a+GFP-Rabin8+miRFP-Rab11a, tRFP-Rab8a+GFP-Rabin8+miRFP-Rab14, cells ± Dox probed with Rabin8 antibody; and RPE WT, tRFP-Ra- b8a+GFP-Rab11a, tRFP-Rab8a+GFP-Rabin8+miRFP-Rab11a cells ± Dox and probed with Rab11a antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was used as a loading control. * corresponds to the endogenous protein detected in RPE WT cells and the arrows indicates the endogenous and exogenous levels in established fluorescent-tagged RPE cells.

Article Snippet: Multisite gateway cloning with LR Clonase II+ (#12538120, Invitrogen) was used to generate BFP-Rab11b and BFP-Rab14 fusion constructs Cell Reports 43, 114955, November 26, 2024 19 cloned into a gateway compatible pCS2+30 vector with an NH2-terminal FLAG-TEV-Stag tag sequence. pENTR Rab11aS25N, Rab11bS25N, Rab5aS34N and Rab14S25N were generated using QuickChange II mutagenesis kit (Catalog no. 200524, Agilent Technologies) and subcloned in pDEST53 to generate GFP-Rab5aSN, GFP-Rab14SN, GFP-Rab11bSN. pENTR Rab11aQ67L, Rab11bQ67L and Rab14Q70L were generated using QuickChange II mutagenesis kit, and along with pENTR Rab11a, Rab11b and Rab14 were subcloned in pDEST-tRFP, a vector previously described in3, to generate tRFP-Rab11a, tRFP-Rab11aQL, tRFPRab11b, tRFP-Rab11bQL, tRFP-Rab14 and tRFP-Rab14QL. mNeonGreen-Rab8a, mCherry-Rab11a, Rabin8 and Rabin8E192A were subcloned into pCS2+ (Addgene, Kit#1000000107) or pCS2-GFP (#86723, Addgene).

Techniques: Western Blot, Control

Journal: Cell reports

Article Title: Rab11-Rab8 cascade dynamics in primary cilia and membrane tubules.

doi: 10.1016/j.celrep.2024.114955

Figure Lengend Snippet: Figure 5. Rabin8 GEF activity is required for Rab8 LTM localization, ciliary trafficking, and ciliogenesis (A) (Right) Quantification of tRFP-positive LTMs in fixed RPE cells stably expressing tRFP-Rab8a and GFP-Rabin8 WT or GEF mutants (E192A and F201A) treated with Dox for 24 h, followed by CytoD for 30 min. (Left) Representative images from a single xy-plane of a z stack captured on an SDCM. Mean ± SEM for 100 cells from n = 3 experiments. (B) Quantification of mNeonGreen-Rab8a and mCherry-Rab11a colocalized on LTMs in yolk sac cells from 24 hfp zebrafish embryos co-injected with Rabin8 or Rabin8 E192A mRNA. Mean ± SEM for n = 10 (Rabin8) and n = 9 (Rabin8 E192A) independent experiments. (C) Quantification of ciliation in cells described in (A) following serum starvation for 24 h and stained with AcTub. Mean ± SEM for 100 cells from n = 3 experiments.

Article Snippet: Multisite gateway cloning with LR Clonase II+ (#12538120, Invitrogen) was used to generate BFP-Rab11b and BFP-Rab14 fusion constructs Cell Reports 43, 114955, November 26, 2024 19 cloned into a gateway compatible pCS2+30 vector with an NH2-terminal FLAG-TEV-Stag tag sequence. pENTR Rab11aS25N, Rab11bS25N, Rab5aS34N and Rab14S25N were generated using QuickChange II mutagenesis kit (Catalog no. 200524, Agilent Technologies) and subcloned in pDEST53 to generate GFP-Rab5aSN, GFP-Rab14SN, GFP-Rab11bSN. pENTR Rab11aQ67L, Rab11bQ67L and Rab14Q70L were generated using QuickChange II mutagenesis kit, and along with pENTR Rab11a, Rab11b and Rab14 were subcloned in pDEST-tRFP, a vector previously described in3, to generate tRFP-Rab11a, tRFP-Rab11aQL, tRFPRab11b, tRFP-Rab11bQL, tRFP-Rab14 and tRFP-Rab14QL. mNeonGreen-Rab8a, mCherry-Rab11a, Rabin8 and Rabin8E192A were subcloned into pCS2+ (Addgene, Kit#1000000107) or pCS2-GFP (#86723, Addgene).

Techniques: Activity Assay, Stable Transfection, Expressing, Injection, Staining