Flp in t rex system

Stable, inducible TRPV4-FLAG 293 cells, referred to as T-Rex-TRPV4 cells, were generated using the Flp-In T-Rex system according to manufacturer protocols (Invitrogen). Briefly, WT TRPV4-FLAG and R269C TRPV4-FLAG in pcDNA3.1 were subcloned into pcDNA5/FRT/TO using the primers listed in Supplementary Table 2. Flp-In T-Rex 293 host cells were then co-transfected with TRPV4-FLAG pcDNA5/FRT/TO and pOG44 flp-recombinase. Stably integrated positive clones were selected with 100 µg/ml hygromycin (Invitrogen) and 15 µg/ml blasticidin (Invitrogen). Final polyclonal isogenic lines were tested for zeocin sensitivity (100 µg/ml) to confirm integration at the FRT site. Cells were cultured in standard media containing DMEM supplemented with 10% FCS. Every 2–3 passages, media was supplemented with 15 µg/ml blasticidin to select against cells that had lost the transgenes. TRPV4-FLAG expression was induced by treatment with 15 ng/ml tetracycline (Invitrogen) for 16–20 h.

Stably transfected human embryonic kidney (HEK293) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 2 mml-glutamine, antibiotics (100 units/ml penicillin, 0.1 mg/ml streptomycin), and in the presence of selection with 100 μg/ml hygromycin and 15 μg/ml blasticidin. Stable HA-tagged CUL5 cells and GFP-tagged TRIAD1 and HHARI cells were described previously (28 (link)). All HA-tagged DCNL1 expression vectors were stably transfected into HEK293 using Invitrogen's Flp-In T-Rex system according to the manufacturer's instructions. The HA–DCNL1 and HA–DCNL1 (DAD^MUT) cells have been described before in Scott et al. (48 (link)). Protein expression was typically induced overnight using 1 μg/ml tetracycline unless indicated otherwise. Where indicated, cells were treated with MLN4924 (Active Biochem) at a final concentration of 3 μm overnight and 2 or 20 μm MG132 (InvivoGen) overnight.

Stable HeLa (ATCC) cell lines inducibly expressing siRNA resistant Kif18A variants were generated using the Flp-In/T-REx system (Invitrogen). Transfection of siRNA targeting human Kif18A and time-lapse microscopy was performed as previously described (Häfner et al., 2014 (link)). High-resolution analysis of mitotic spindles was performed as previously described in Mayr et al. (2007) (link).

The SH-SY5Y-derived FIp-In host cells [28] (link) and Flp-In T-REx System (Invitrogen) was used to generate stably induced SH-SY5Y cell lines exhibiting tetracycline-inducible expression of Tyr52 and Cys52 FBXO7. Briefly, the SH-SY5Y host cells were co-transfected with pOG44 plasmid (constitutively expressed the Flp recombinase) and pcDNA5/FRT/TO-FBXO7-EGFP plasmid according to the supplier's instructions. These cell lines were grown in medium containing 5 µg/ml blasticidin and 100 µg/ml hygromycin. Doxycycline (dox, 5 µg/ml) was added to induce EGFP-tagged FBXO7 expression for two days. The proteins were prepared for Western blotting using antibody to FBXO7 or actin as described. Neuronal phenotypes were examined after induced differentiation with retinoid acid (10 µM) and induced expression of FBXO7 for 7 to 21 days. The morphologic differentiation of Tyr52 and Cys52 SH-SY5Y cells including total outgrowth, processes, and branches was assessed by using Metamorph microscopy automation and image analysis software (Molecular Devices).

GFP-, GFP-TIA1b-, GFP-TIARb-, and GFP-HuR-expressing FT293 cell lines were generated using the Flp-In T-Rex System (Invitrogen) [17 (link)]. Immunocytochemistry was carried out as described [16 (link), 17 (link)]. Murine embryonic fibroblasts (MEFs) knocked-out for TIA1 [26 (link)], TIAR [27 (link)], or EIF2AK2 [32 (link)] were maintained as described [33 (link)]. The lentiviral particles were generated from plasmids of second generation (pLKO series) that were transfected into HEK293T and the supernatants were collected. The shRNA against the human EIF2AK2 gene comes from SIGMA MISSION (# TRCN0000196400).