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Nat. but not cilia formation. Together, these two TULP domains play unique tasks in ciliary protein trafficking but are insufficient for cilia formation in RPE-1 cells. In addition, TULP1 and TULP2 play additional unfamiliar molecular tasks that should be resolved in the future. tubby homolog dTULP and the homolog tub-1 are also involved in the ciliary trafficking of membrane receptors (DiTirro et al., 2019; Park et al., 2013). mice show Xipamide maturity-onset obesity with neurosensory deficits (Kleyn et al., 1996; Noben-Trauth et al., 1996), both common ciliopathy characteristics. Although the particular cell types and GPCRs that cause the obesity phenotype of mice have not yet been recognized, the phenotype is almost certainly linked to TUBs ciliary functions. On the other hand, TULP3 was originally identified as Rabbit polyclonal to AMHR2 a negative regulator of Hedgehog signaling (Norman et al., 2009), and Xipamide the TULP3-dependent nature of ciliary GPCR trafficking has been clearly implicated in the underlying mechanism (Mukhopadhyay et al., 2010). While some of the molecular functions of mammalian TULPs, especially of TULP3 and TUB, have been recognized, those of TULP1 and TULP2 have not. Whereas the loss of TULP1 function prospects to retinal degeneration in Xipamide both humans and mice (Hagstrom et al., 1999; Ikeda et al., 2000), presently there are not yet any functional data available for TULP2. Because Xipamide TULP1 and TULP2 each have either the conserved IFT-A-binding domain name or the membrane phosphoinositide-binding domains of the TULPs, and because these domains are required for the ciliary trafficking of GPCRs, there is a good chance TULP1 and TULP2 share some of the molecular functions of TULP3 and TUB. Here, we statement the results of our investigation into the ciliary functions of the four TULPsTULP1, TULP2, TULP3, and TUBin hTERT RPE-1 (RPE1) cells. We found that, despite significant amino acid-level similarity, these four proteins play individual functions in main cilia assembly and trafficking. The functions of TULP3 and TUB are largely analogous with respect to cilia formation and ciliary protein trafficking. In contrast, although TULP1 and TULP2 show limited capacity to control ciliary protein trafficking, the addition of an IFT-A binding domain name increases it. Together, while TULP3 and TUB are crucial ciliary membrane trafficking regulators, TULP1 and TULP2 must play different molecular functions that should be investigated in the future. MATERIALS AND METHODS Cell culture Both RPE1 wild-type (CRL-4000; American Type Cell Collection [ATCC], USA) and knockout (KO) cells were cultured in DMEM/F-12 (11320082; Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS) (10099141; Thermo Fisher Scientific) and 1% Penicillin/Streptomycin (P/S) (15-140-122; Thermo Fisher Scientific). Cells were produced on coverslips in 12-well culture plates at a density of 0.5 106 cells/well and cultured in a CO2 incubator. Cells were shifted from 10% to 0.5% serum 24 h after transfection to induce ciliation and fixed 72 h post-transfection. Plasmid transfections were carried out with a NEPA21 Electroporator (NEPA GENE, Japan) at a pulse voltage of 150 V and pulse length Xipamide of 5 ms. RNA isolation and quantitative RT-PCR Total RNA was extracted using the RNeasy mini kit (74104; Qiagen, Germany). Complementary DNA (cDNA) was synthesized from total RNA with the RevertAid Reverse Transcriptase (EP0442; Thermo Fisher Scientific) in accordance with the manufacturers.

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