MCF-7 cells were transfected with the control vector pcDNA 3.1 (+/hygromycin) (Invitrogen, Carlsbad, CA, USA), or pcDNA 3.1-PRB using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Stable transfected cells were selected in DMEM containing hygromycin B at 400 µg/mL. Selection of positive clones was based on PRB protein expression by Western Blot analysis. Validation was performed for cells in three consecutive passages before experiments with stable clones were started.
Pcdna3.1 hygromycin
Manufactured by Thermo Fisher Scientific
Sourced in United States
PcDNA3.1/hygromycin is a plasmid vector used for mammalian cell expression. It contains a hygromycin resistance gene for selection of stable transfectants.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Phospho total erk 1 2 whole cell lysate kit, by Mesoscale (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Galanthus nivalis lectin agarose, by Vector Laboratories (1 mentions)
Hcas9, by Addgene (1 mentions)
Gibson assembly, by New England Biolabs (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
6 protocols using pcdna3.1 hygromycin
1
Stable Cell Line Generation for PRB
2
Measuring FGFR and β-Klotho Signaling
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L6 rat myoblast cells were maintained in Dulbecco's modified Eagle's medium (Mediatech) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) and 1× penicillin/streptomycin (Invitrogen) in a 37 °C incubator with 5% CO2. Cells were plated in 96-well plates, and 24 h post plating, cells were transfected with expression vectors encoding human β-Klotho and individual human FGFR isoforms using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. FGFR and β-Klotho were previously cloned into the pcDNA3.1(+)-hygromycin (Invitrogen) and pTT14 vectors, respectively. Cells were serum-starved in Dulbecco's modified Eagle's medium supplemented with 0.2% (w/v) BSA overnight the day after transfection and treated with vehicle (PBS) or test molecules for 10 min. Following treatment, the medium was aspirated and cells were snap frozen in liquid nitrogen. Cell lysates were prepared and total ERK and pERK levels were measured with the Phospho/Total ERK1/2 Whole Cell Lysate Kit (Meso Scale Discovery) according to the manufacturer's instructions. All experiments were run in duplicate.
3
Production and Characterization of HIV-1 Env Proteins
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HIV-1 Envs for ELISA and SPR assays included HIV-1 MN recombinant gp41 (Immunodiagnostics), HIV-1 group M consensus gp120 (CON-S) (Liao et al., 2006 (link)), HIV-1 clade C consensus (ConC) gp120, ConC gp120 N332A mutant, Env immunodominant region peptide sp400 (RVLAVERYLRD-QQLLGIWGCSG-KLICTTAVPWN-ASWSNKSLNK), gp41 MPER region peptide SP62 (QQEKNEQELLELDKWASLWN) and GCN4 gp41 Inter (Frey et al., 2010 (link)). CAP206 autologous transmitted/founder env and 6 additional envs from the first 30 months of infection were obtained from serial blood samples by single genome amplification (Keele et al., 2008 (link)), codon optimized (Andre et al., 1998 (link)) and de novo synthesized (GeneScript) as gp140 or gp120, and cloned into pcDNA3.1/hygromycin (Invitrogen). Recombinant Env glycoproteins were produced in 293F cells in serum-free media transfected with HIV-1 gp140 or gp120 expressing pcDNA3.1 plasmids, purified from the supernatant of transfected 293F cells using Galanthus nivalis lectin-agarose (Vector Labs) column chromatography, and stored at − 80 °C.ELISA was performed as described (Liao et al., 2011 (link), Liao et al., 2013a (link)).
4
Generating Targeted FBH1 Knockout Cell Lines
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We synthesized gRNAs flanking exon 4 and 5 and cloned them into the gRNA cloning vector (Addgene # 41824) using Gibson assembly (New England Biolabs)[19 (link)]. The targeting sequences for gRNAs were: FBH1 gRNA-1 5’-GCGGGGCACTTTGTGAGTGG-3’ and FBH1 gRNA-2 5’-GCGGCATGTTATTTCACTGG-3’. Cells were co-transfected with FBH1 gRNA-1, FBH1 gRNA-2, hCas9 (Addgene# 41815) and pcDNA 3.1 Hygromycin (Invitrogen) using Lipofectamine 3000 as per manufacturers’ instructions. Single colonies were isolated after selection in 100 ug/ml hygromycin. After growing colonies to a 96-well plate, clones were split into two 96-well plates. Genomic DNA was isolated from one well using QuickExtract solution following manufacturers’ protocol. Clones were screened for deletion of Exons 3 and 4 by PCR using Phusion polymerase. Primers used in screening: FBH1_F 5’-CCTGATGACCCCAAGCATG-3’, FBH1_R1 5’-GGTCCCGGTAGCCTGGTTAC-3’, and FBH1_R2, 5’-AAGATTCCTCAACCAAATGG-3’. Clones positive by PCR were expanded and screened for loss of FBH1 by Western blotting.
5
hIL-10 cDNA Cloning and Expression
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Human interleukin-10 (hIL-10) complementary DNAs (cDNA) were synthesized by reverse transcription-polymerase chain reaction (RT-PCR) from mRNA of THP-1 cells using the PCR cloning primers. The PCR product was inserted to pcDNA3.1/hygromycin (−) (Invitrogen) with restriction enzymes, NotⅠ and BamHⅠ.
6
Codon-optimized Transmitted/Founder Env Genes
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