The pcDNA3.1(−)-TIM-1 and pVAX1-NS1′ were preserved in our laboratory. JEV E gene was inserted into p3×FLAG-CMV-7.1. hTIM-D114A-His, hTIM-N115A-His, and hTIM-MD were cloned into pcDNA3.1 (+). hTIM-IgVD was cloned into pCAGGS. hTIM-ECD was inserted into prokaryotic expression vectors pET-28a (+) and pGEX. Mouse anti E monoclonal antibody was kept by our lab. Mouse anti-human TIM-1 monoclonal antibody (MAB1750, R&D systems, Minneapolis, MN, USA) and goat anti-TIM-1 polyclonal antibody (AF1750, R&D systems, Minneapolis, MN, USA) were used for detecting the TIM-1 protein in Western blot and IFA. Mouse anti-His antibody (66005-1-Ig, Proteintech, Rosemont, IL, USA) was used for the enrichment of TIM-1-His protein. Mouse anti-GAPDH polyclonal antibody (sc-25778, Santa Cruz, Dallas, TX, USA) was used for Western blot. Rabbit anti-JEV E polyclonal antibody (GTX125867, GeneTex, Irvine, CA, USA), rabbit anti-JEV NS3 polyclonal antibody (GTX125868, GeneTex, Irvine, CA, USA), and rabbit anti-JEV polyclonal prM antibody (GTX131833, GeneTex, Irvine, CA, USA) were used to detected the JEV E, NS3 and prM protein, respectively, in Western blot analyses. Donkey anti-goat IgG-Alexa Fluor488 (ab150129, Abcam, Cambridge, UK), donkey anti-rabbit IgG Alexa Flour 594 (ab150076, Abcam, Cambridge, UK), and donkey anti-mouse Alexa Flour647 (ab150107, Abcam, Cambridge, UK) were used for IFA.
Af1750
Manufactured by R&D Systems
Sourced in United States
The AF1750 is a laboratory equipment product. It is designed to perform a specific core function. No further details about the intended use or interpretation of the product's capabilities are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab150129, by Abcam (1 mentions)
Ab150076, by Abcam (1 mentions)
Ab150107, by Abcam (1 mentions)
Mab1750, by R&D Systems (1 mentions)
Mouse anti gapdh polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Gtx125868, by GeneTex (1 mentions)
Gtx125867, by GeneTex (1 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Trizol, by Takara Bio (1 mentions)
Baf007, by R&D Systems (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Streptavidin pe, by Thermo Fisher Scientific (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Streptavidin sepharose bead, by GE Healthcare (1 mentions)
Mab97792, by R&D Systems (1 mentions)
Af154, by R&D Systems (1 mentions)
Novocyte quanteon cytometer, by Agilent Technologies (1 mentions)
8 protocols using af1750
1
Plasmid Constructs and Antibodies for JEV
2
TIM-1 Expression and Antibody Production
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Total RNA was extracted from A549 cells by Trizol (TaKaRa Bio, Kyoto, Japan), and then it was reverse transcribed to produce cDNA using PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa). The full-length sequence of the gene encoding TIM-1 was amplified by polymerase chain reaction (PCR) from A549 cells. The TIM-1 gene open reading frame (ORF) was cloned into pcDNA3.1 (−) using BamHI and XbaI restriction sites. The ORF of TIM-1 was amplified with oligos 5′-GCTCTAGAATGCATCCTCAAGTGG-3′ and 5′-ATGGATCCTTAGTCCGTGGC-3′. Specific monoclonal antibodies for JEV E and NS1 were produced in our lab, and an NS5 monoclonal antibody was purchased from Gene Tex (Irvine, CA, USA). A GAPDH polyclonal antibody (sc-25778) was purchased from SantaCruz (Dallas, TX, USA). A TIM-1 monoclonal antibody (MAB1750) and polyclonal antibody (AF1750) were purchased from R&D systems.
3
Colloidal Gold-Based Immunochromatographic Assay for KIM-1 Detection
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The immunochromatographic strip consisted of five sections, which were sourced from Shanghai Goldbio Technology Co., Ltd., unless stated otherwise. The section consisted of the following components: A sample pad fiberglass membrane (SB06), a conjugate pad polyester fiber membrane (VL78), an analyzing nitrocellulose membrane (Merck Millipore), an absorbent pad (CH27) and a plastic back plate. The conjugate pad was pretreated with the purified colloidal gold-McAb KIM-1 conjugates, and then dried in an incubator at 37°C for 30 min. A control (C) line and test (T) line were marked on the analyzing membrane with goat anti-mouse IgG (2 mg/ml; BAF007, R&D Systems, Inc.) and goat anti-human PcAb KIM-1 (200 µg/ml, dilution, 1:200, AF1750, R&D Systems, Inc.), respectively. The sample pad, pretreated conjugate pad, analyzing membrane and absorbent pad were pasted on a plastic back plate. The detection limit of the colloidal gold-based immunochromatographic strip was determined by detecting normal urine specimens supplemented with KIM-1 at the final concentrations of 0, 50 and 100 ng/ml, and 1 and 10 µg/ml, respectively.
4
Zika Virus Infection Imaging Protocol
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Duplicate well of cells was infected with ZIKV as above and processed for imaging at 72 and 144 hpi. Cells were washed, fixed with 10% neutral buffered formalin, permeabilized with 0.3% Triton X-100 in PBS for 20 min, and blocked with 5% BSA in PBS overnight at 4°. Then cells were stained with anti-flavivirus E protein antibody 4G2 conjugated to AF647 (Novus biologicals catalog NBP2-52709AF647) at 1:500 for a minimum of 1 h. After washing, cells were counterstained with DAPI and imaged using an EVOS FL cell imaging system (ThermoFisher). DAPI stained nuclei were counted using the automatic cell counting feature in the EVOs software (software revision 32,044), and 4G2 positive cells in the same fields were counted by hand. At least 10 fields and 1,000 DAPI positive cells were counted for each well. For receptor transfection experiments, DC-SIGN transfected cells were stained with a mouse antibody against DC-SIGN conjugated to AF647 (Biolegend 33011). TIM-1 transfected cells were stained with goat-anti-TIM-1 antibody (R&D systems AF1750), then with an anti-goat secondary antibody conjugated to AF488 (Invitrogen A-11055). Cells were counterstained with DAPI and imaged on an EVOS FL system as above.
5
Quantification of Plasma KIM-1 Levels
6
Quantification of hTIM-1 Surface Expression
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HEK293T cells were seeded in 6-well plates (2.5 × 104 cells per well) precoated with poly-L-lysine (Cultrex, R&D Systems, Minneapolis, MN, USA) and incubated overnight at 37 °C. The cells were transfected with pCAGGS encoding WT hTIM-1 or its SNV mutant genes. At 24 h post-transfection, the cells were washed with PBS, detached by treatment with 0.25% trypsin, and then fixed with 4% paraformaldehyde in PBS for 15 min. After washing with PBS, the cells were incubated with a goat anti-hTIM-1 polyclonal antibody (AF1750, R&D Systems, Minneapolis, MN, USA) for 1 h at room temperature. Then the cells were washed with PBST and stained with a donkey anti-goat IgG polyclonal antibody conjugated with FITC (sc2024, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 4 °C in the dark. After washing 3 times with PBST, the percentage of FITC-positive cells and mean fluorescent intensity (MFI) of FITC signals were analyzed using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo software (Tree Star, San Carlos, CA, USA).
7
Biotinylation of Surface Proteins in Viral Infected Cells
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HAP1 and HAP1ΔCDC50 cells were plated in a 6-well plate at a density of 1×106 cells/well. Cells were either mock infected or infected one day after plating with CHIKV-GFP (MOI 0.5) or LCMV-GFP (MOI 1) and harvested at the indicated time points. Cells were washed with cold PBS, and surface proteins were biotinylated with 0.5 mg/mL sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate (ThermoFisher) on ice for 45 minutes with gentle shaking. The reaction was quenched using Tris-HCl and cells were lysed with M2 lysis buffer at 4°C. Samples were centrifuged at 17,000xg for 10 minutes. A fraction of the lysate was saved (total cell lysate, TCL), and the surface proteins were bound to streptavidin Sepharose beads overnight at 4°C (GE Healthcare). Beads were then washed with buffer containing 100 mM Tris, 500 mM lithium chloride, 0.1% Triton X-100 followed by a buffer containing 20 mM HEPES [pH 7.2], 2 mM EGTA, 10 mM magnesium chloride, 0.1% Triton X-100. Samples were then analyzed through immunoblotting probing against TYRO3 (1:1000, R&D Systems, MAB859100), TIM (TIM (1:500, AF1750, R&D Systems), AXL (1:100, AF154, R&D Systems), GAPDH (1:2000, Santa Cruz Biotech, #sc-47724), Transferrin (1:1,000, PA5–27739, ThermoFisher), or CHIKV E1 (1:1,000, MAB97792, R&D systems).
8
CHIKV-GFP Infection and Cell Surface Marker Profiling
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Cells were plated at a density of 1.0×106 cells/well in a 6-well plate one day prior to staining. Cells were harvested either uninfected or after infection with CHIKV-GFP at an MOI of 0.5, 18 hours post-infection. Monolayers were cooled, washed, and treated with a blocking solution (dPBS +Ca2 +Mg2 with 2% (v/v) FBS) containing an anti-hTIM1(1:50–1:100, AF1750, R&D Systems) antibody. Samples were incubated at 4°C with gentle shaking for one hour and washed three times with ice-cold PBS. A blocking solution containing the corresponding secondary antibody, donkey anti-goat Alexa Fluor 594 (1:2500, A32758, Invitrogen), was added. Samples were incubated at 4°C in the dark with gentle shaking for 30 minutes. Samples were washed three additional times with PBS, lifted via scraping, and analyzed using a NovoCyte Quanteon cytometer (Agilent). Populations of live, single cells were gated using FSC/SSC and SSC-A/SSC-H, respectively. The GFP gate was set using uninfected, GFP- cells, and the AF594 gate was set with a secondary-only control. The AF594 MFI of 10,000 live, single, GFP+ cells was quantified. A 488-nm laser with a 530/30 “FITC” bandpass filter was used to assess GFP fluorescence, and AF594 was measured with a 561-nm laser with a 615/20 “PE-Texas Red” bandpass filter; all filter sets had default gain.
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