Amicon ultra 15 ml centrifugal filter

Supernatants of liquid cultures were obtained by filtration through a double layer of Miracloth (Merck Millipore, Burlington, MA, United States). Fractionation of supernatants was performed using Amicon Ultra 15 ml centrifugal filters (Merck Millipore, Burlington, MA, United States) with molecular weight cut-offs of 3, 10, 30, and 100 kDa, respectively, according to manufacturer’s recommendations.

To collect conditioned media, 1 × 10⁶ cells were seeded on 10 cm diameter plates. The next day, cells began to be exposed to culture medium supplemented with one or more drugs, with replacement of the medium with the drug(s) after 48 h (this to ensure the maximum activity of the drug(s) during the entire exposure time). After 96 h of exposure, cells were washed three times in 1×PBS. After washing, 4 mL of serum-free medium was added to the plates. Then, 24 h later, conditioned media were collected in 2 mL Eppendorf tubes. Conditioned media were centrifuged for 5 min at 1000 rpm and 4 °C, and then stored in new tubes at −80 °C. After collecting ~25 mL per experimental condition, conditioned media were cold-thawed and concentrated 30-fold using Amicon Ultra 15 mL centrifugal filters (Merck Millipore Ltd., Tuliagreen, Carrigtwohill, Co Cork, Ireland). Aliquots of 50 μL of concentrated medium were stored at −80 °C until use.

To reduce the safety risk of aerosolized virus, a pseudo-typed virus with the SARS-CoV-2 Spike protein expressed on the envelop surface carrying a green fluorescent protein (GFP) reporter gene was chosen as the test virus, which was produced in HEK293T cells according to the manufacturer’s instructions (InvivoGen, Hong Kong, China, cat. no. PLV-SPIKE)^{27 (link)}. Briefly, 70–80% confluent HEK293T cells were transfected with envelope plasmid pLV-Spike, packaging plasmid and transfer plasmid carrying a GFP reporter gene with LentiTran transfection reagent (OriGene, Rockville, MD). 1-day post-transfection, the culture medium was removed, and the cells were washed with fresh medium twice to prevent plasmid DNA carryover. Cell media containing pseudo-typed viruses were then collected daily for 3 days. After collection, pseudo-typed virus containing media were centrifuged at 1000×g for 10 min and filtered through a 0.45-µm Polyethersulfone (PES) syringe filter (Thermo Fisher Scientific, Rockford, IL) to remove cell debris. The pseudo-typed viruses in the filtrate were then concentrated with Amicon® Ultra 15 mL Centrifugal Filters, 100 kDa nominal molecular weight limit (NMWL) (Merck Millipore, Darmstadt, Germany) before aliquoting and stored at − 80 °C before use.

AnkX was used to quantitatively transfer PC-Cl to 10x-His-tagged-Rab1b from the cosubstrate CDP-Choline-Cl. Quantitative modification of Rab1b with the phosphocholine derivative was confirmed using intact MS. After modification with PC-Cl, the SEC-purified binary adduct Rab1b_S76(PC-Cl) was co-incubated with equimolar amounts of Lem3_21-486 over night at 19 °C. Complex formation was confirmed using SDS-PAGE gel shift assay and 25 mM imidazole were added prior to loading on a 5 ml Nuvia IMAC column (Bio-Rad Laboratories). The column was washed with 50 mM imidazole for 400 ml to remove free Lem3. The complex and free Rab1b were eluted with 125 mM imidazole and dialysed against dialysis buffer_Rab1b overnight in presence of PreScission protease. SEC was performed to separate the complex from free Rab1b and oligomeric species. The complex was concentrated to desired concentration using Amicon Ultra 15 ml centrifugal filters (Merck Millipore).

Recombinant human TRAP 5a or TRAP isoforms derived from cell lysates were separated as described previously [38 , 50 (link)]. Briefly, an ÄKTA-purifier™ 10 FPLC system (GE Healthcare, Sweden) was applied with a Heparin column (Pharmacia) at a flow rate of 2 mL/ min at 4 °C. The Heparin column was equilibrated with 20 mM Tris-HCl (pH 7.2), 0.1 M NaCl, 0.005% Triton X-100 (w/v) and the protein in lysis buffer or medium eluted with a linear gradient of NaCl from 0.1 M – 1 M in 20 mM Tris-HCl (pH 7.2), 0.005% Triton X-100 (w/v) and collected in fractions of 1 mL each. FPLC spectra were compiled by measuring TRAP enzymatic activity per fraction and fractions further pooled and concentrated with Amicon® Ultra 15 mL Centrifugal Filters (MerckMillipore) for protein expression studies.