Flp in t rex 293 cell

All cell lines were purchased from the American Type Culture Collection (ATCC; www.atcc.org) and cultured according to standard mammalian tissue culture protocols at 37 °C, 5% CO₂ in a humidified incubator. NIH-3T3 cells were cultured in DMEM (Hyclone, SH30243.01) supplemented with 10% bovine serum (Invitrogen, 16170-078) and antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin). HepG2 cells were cultured in DMEM (Hyclone, SH30243.01) supplemented with 10% fetal bovine serum (Gibco, 16000-044), 1% GlutaMax (Gibco, 35050061) and antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin). AML12 cells were cultured in DMEM/F12 (Gibco, 11320-033) supplemented with 10% FBS, 1% Insulin-Transferrin-Selenium (Gibco, 41400-045) and antibiotics. 293AD cells and HeLa cells were cultured in DMEM supplemented with 10% FBS and antibiotics. Flp-In T-REx 293 cells (Invitrogen, R78007) were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics. For transient plasmid transfection, cells were plated at 2.5 × 10⁵ cells/well in a 6-well culture plate. Twenty-four hours after plating, cells were transfected using 6 μL jetPEI (Polyplus) and 2.5 μg GFP, TurboID-KDEL, or Sec61b-TurboID plasmids according to manufacturer protocols.

The inducible pcDNA5 FRT/TO FLAG×3-HA×3-K-Rta expression construct was first generated by cloning of 3×FLAG and 3×HA tandem epitope tags fused to the N-terminal region of K-Rta. Inducible 293 cells were subsequently generated by cotransfection of pcDNA5 FRT/TO FLAG×3-HA×3-K-Rta and pOG44 (Flp recombinase) into Flp-In TREx-293 cells following the manufacturer's protocol (Invitrogen). The K-Rta-inducible 293 cells were then used to create cells stably expressing His-ubiquitin by transfection of pcDNA-His-ubiquitin. Stable clones were selected in the presence of zeocin for 3 weeks. Several clones were picked, and His-ubiquitin expression in the presence or absence of MG132 (10 μg/ml) was confirmed. Multiple clones were then pooled and used for these studies.

All combinations of the Y511A mutation were stably expressed in Flp-in TREX 293 cells following manufacturer’s protocol (Invitrogen, MA, USA). Briefly, cells were co-transfected (performed as described above) with pcDNA5/FRT/TO containing the gene of interest and the Flp-recombinase expression vector pOG44 into the Flp-In host cell line (Invitrogen, MA, USA) at a ratio of 1:9, respectively, as previously described12 (link). Successful recombination and maintenance of the gene of interest was confirmed through hygromycin B (200 μg/ml; Sigma-Aldrich, MO, USA) selection to establish a stably-transfected cell line.

Flp-In T-REx 293 cells (Invitrogen) were cultured with DMEM supplemented with 10 μg/ml blasticidin. pOG44 and pcDNA5/FRT/TO_Stx5-opsin plasmids were co-transfected. At 2 days after transfection, cells were selected by incubating with DMEM++ supplemented with 15 μg/ml blasticidin and 200 μg/ml hygromycin B for 2 weeks until obtaining positive clones. The expression of Stx5–op from the stable transfectants was tested by western blotting. For inducing the expression of Stx5–op, 1 μg/ml tetracycline was added into the medium and the cells were incubated at 37°C for 6 h.

Flp-In T-REx 293 cells (Invitrogen) were transfected with a pcDNA5/FRT/TO plasmid expressing WT B14-3xFLAG along with pOG44 plasmid expressing a Flp recombinase. Selection was carried out with media containing hygromycin and blasticidin over several weeks. Expression of B14-3xFLAG to near endogenous levels was induced with 5 ng/mL of tetracycline provided to the media for 16 h. Three confluent 15 cm plates were collected in PBS and lysed for 30 mins on ice in 2.5 mL of buffer containing 0.1% digitonin, 50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA and protease inhibitor. Lysate was cleared with centrifugation at 20,000g for 15 min. Lysate divided in half was incubated for 2 h at 4°C with 30 μL of anti-FLAG M2 agarose beads that was pre-incubated with or without 3xFLAG peptide (100 μL, 0.25 mg/mL). Agarose beads were washed extensively with buffer lacking detergent. Bound proteins were eluted overnight at 4°C with 3xFLAG peptide (200 μL, 0.25 mg/mL). Three subsequent elutions were performed for 1 h each, pooled and concentrated using centrifugal filters (Amicon Ultra 0.5 mL 3K membrane). SDS sample buffer was added and heated for 30 min at 37°C followed by SDS-PAGE and silver staining or immunoblotting. Bands excised from a silver stained gel were analyzed by mass spectrometry at Taplin Biological Mass Spectrometry Facility (Harvard Medical School).