Expression constructs for mouse and human Wnt proteins with various N-terminal tags were designed to include tobacco etch virus (TEV) protease cleavage sequence after the tag as shown in Supplementary file 1 , and constructed by extension PCR. Expression plasmids for various albumin family proteins with N-terminal tag (either Tg or PA tag) were also constructed by the same strategy. The coding region for Tg-Wnt3a was subcloned into a mammalian episomal expression vector pEBMulti-Hyg (Wako Pure Chemical, Japan) and used to transfect HEK293S GnT1- cells. Transfected cells were selected against medium containing 0.2 mg/ml hygromycin B, and the resistant polyclonal cell population confirmed to maintain high Wnt3a-producing ability over the repeated passages were used for the protein production. HEK293S GnT1- cells stably producing Tg-Wnt5a were established in a similar manner. For the co-expression of Wnt and AFM, expression plasmids for Tg-hAFM and N-terminally PA tagged mouse or human Wnts were co-transfected at a DNA ratio of 10:1 using the Expi293 Expression System (Life Technologies Inc. Tokyo Japan), according to the procedures recommended by the manufacturer. The culture medium was harvested at 90 hr post-transfection, and immediately used for the Wnt/β-catenin reporter assay or the protein purification.
Pebmulti hyg
Manufactured by Fujifilm
Sourced in Japan
The PEBMulti-Hyg is a laboratory equipment product offered by Fujifilm. It is designed for thermal processing applications. The core function of the PEBMulti-Hyg is to provide controlled heating and cooling capabilities for various laboratory processes.
Automatically generated - may contain errors
Lab products found in correlation
Viafect transfection reagent, by Promega (2 mentions)
Hygromycin b solution, by Nacalai Tesque (2 mentions)
Expi293 expression system, by Thermo Fisher Scientific (1 mentions)
Pgl3 luciferase reporter vector, by Promega (1 mentions)
Pcdf1 mcs2 ef1 puro lentiviral expression vector, by System Biosciences (1 mentions)
Gibson assembly master mix, by New England Biolabs (1 mentions)
C 22010, by PromoCell (1 mentions)
C 12203, by PromoCell (1 mentions)
Human umbilical vein endothelial cells (huvec), by PromoCell (1 mentions)
Endothelial cell growth medium, by PromoCell (1 mentions)
Pgex 6p 1, by GE Healthcare (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Pgl3 promoter vector, by Promega (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
7 protocols using pebmulti hyg
1
Recombinant Expression of Wnt Proteins
2
Cloning and Luciferase Reporters for Fgf21
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Fv2E-Perk and Atf4 were cloned into the pCDF1-MCS2-EF1-Puro lentiviral expression vector (System Biosciences), pEBMulti-Hyg (Wako Pure Chemicals), and pcDNA3.1 (Thermo Fisher–Life Technologies). Regions upstream of the murine Fgf21 transcription initiation site (−1326 to +100, −950 to +100, and −110 to +100) were cloned into pGL3 luciferase reporter vectors (Promega). PCR-based site-directed mutagenesis was used to generate AARE1 and -2 mutants. The DNA sequence of each construct was verified on an ABI 3130 DNA sequencer (Thermo Fisher–Applied Biosystems).
3
Plasmid Construction for AcrIIA4 Expression
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Plasmid DNA encoding AcrIIA4 and FLAG tag genes was synthesized by Eurofins Genomics. The pFucci-G1 Orange Expression vector was purchased from MBL. The gRNA Cloning Vector and SpyCas9 were gifts from George Church (Addgene plasmids # 41824 and 41815). The AcrIIA4 fragment was produced by digesting amplified DNA with BamHI and BstXI. This fragment was ligated into the pFucci-G1 Orange expression vector at the N-terminus of hCdt1(30–120) to construct AcrIIA4-hCdt1(30–120) plasmid DNA. The AcrIIA4 fragment was amplified by CMV primer and Acr-REsite_XbaI_Rv and digested by BamHI and XbaI. This fragment was ligated into the pFucci-G1 Orange vector, which was digested by BamHI and XbaI to construct AcrIIA4 plasmid DNA. To introduce the NLS, the primers BamHI_NLS-AcrIIA4_Fw and Acr-REsite_XbaI_Rv were used. DNA fragment was amplified and digested by BamHI and XbaI, then inserted into the original vector, which was digested by BamHI and XbaI. New plasmid DNAs encoding AcrIIA4-2A-Cas9 or AcrIIA4-Cdt1-2A-Cas9 were constructed using Gibson Assembly. AcrIIA4, AcrIIA4-Cdt1, and SpyCas9 fragments were amplified by PCR. NotI-treated pEBMulti-Hyg (FUJIFILM Wako) and each fragment were inserted into the pEB vector using the Gibson Assembly Master Mix (NEB). All primer sequences are shown in Supplementary Table 1 .
4
Investigating RasGRP2 Effects on HUVEC Barrier
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The TERT HUVEC were kindly provided by Dr. Kazuto Nishio (Kindai University). HUVEC (C-12203; PromoCell) and TERT HUVEC were grown in endothelial cell growth medium (C-22010; PromoCell) under standard cell culture conditions (humidified atmosphere, 5% CO2, 37 °C). Stable cell lines were prepared as previously described23 (link). Briefly, the DNA fragment of rasgrp2 was amplified from human placenta cDNA (K1420-1; Clontech) using the Expand Long Range dNTPack (4829042001; Sigma-Aldrich), pEB Multi-Hyg (050-08121; FUJIFILM Wako Pure Chemical Corporation) was used as a vector, and ViaFect Transfection Reagent (E4981; Promega) was used as the transfection reagent. Cells were seeded in a 24-well plate, grown overnight, transfected, and purified to prepare TERT HUVEC R and mock cell lines. Transfected cells were purified with 50 μg/mL hygromycin B solution (09287-84; Nacalai Tesque Inc.).
Cells were then seeded (0.6 × 105 cells/mL) in culture dishes or cell culture inserts and incubated for six days (medium changed every second day) before experiments. TEER measurements on barrier formation were carried out during this time. Cells were pre-incubated with or without 5 mM NAC, 20 μM DPI, 30 μM apocynin, or 10 μM LY for 2 h, and incubated with 50 μg/mL BSA or TAGE for 24–48 h to investigate the specific effects of RasGRP2.
Cells were then seeded (0.6 × 105 cells/mL) in culture dishes or cell culture inserts and incubated for six days (medium changed every second day) before experiments. TEER measurements on barrier formation were carried out during this time. Cells were pre-incubated with or without 5 mM NAC, 20 μM DPI, 30 μM apocynin, or 10 μM LY for 2 h, and incubated with 50 μg/mL BSA or TAGE for 24–48 h to investigate the specific effects of RasGRP2.
5
Generating TERT HUVEC Cell Lines
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Cells were maintained in the medium using Endothelial Cell Growth Medium (PromoCell, Heidelberg, Germany). pEB Multi-Hyg (Wako Pure Chemicals, Osaka, Japan) was used as vector to prepare TERT HUVEC R and mock cell lines. The DNA fragment of rasgrp2 was isolated by the method previously described [9 (link)]. ViaFect™ Transfection Reagent (Promega, Madison, WI, USA) was used as the transfection reagent. Cells at the concentration of 0.5 × 105 cells/mL were seeded in a 24-well plate, grown overnight, and transfected. Transfected cells were purified with 50 μg/mL Hygromycin B solution (Nacalai Tesque Inc, Tokyo, Japan).
6
Cloning and Expression of IER5 and HSF1
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The coding region of IER5 was cloned into the gene expression vectors pEBMulti-Hyg (Wako Pure Chemicals, Osaka, Japan) and pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA) to create pEBMulti-IER5 and pcDNA-IER5, respectively [27] . For the expression of an influenza hemagglutinin (HA)-tagged protein, the coding region was cloned into pBK266, a derivative of pcDNA3.1(+) containing three copies of the HA-tag sequences (pcDNA-IER5-HA) [28] . Plasmid pK732 is a derivative of pcDNA3.1(+) containing HSF1-HA [29] . Nucleotide substitution mutations were introduced by standard methods. The HSF1 fragment was also cloned into the glutathione S-transferase (GST) gene fusion vector pGEX-6P-1 (GE Healthcare, Buckinghamshire, UK). The cDNA of B55 family members was amplified by RT-PCR from the total RNA of HeLa cells and cloned into pcDNA3.1(+), pBK266, and pGEX-6P-1. The firefly reporter construct HSE-SV40-LUC contained HSE4P oligonucleotides upstream of the SV40 promoter of the pGL3-Promoter vector (Promega, Madison, WI, USA) [27] . The firefly reporter construct HSP70-LUC contained the promoter region of HSPA1A in the pGL3-Basic vector (Promega) [27] .
7
Silencing HSF1 and GADD45β in HeLa Cells
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A plasmid containing a HSF1 shRNA expression construct and a puromycin-resistance gene was introduced into HeLa cells [19, 20] . Cells were cultured in medium supplemented with 1 µg/ml puromycin to select HSF1-silenced cells. For silencing GADD45β, cells were transfected with GADD45β siRNA [21] . The coding regions of GADD45α, GADD45α1, GADD45β, and GADD45γ were amplified by RT-PCR from HeLa total RNA. The influenza hemagglutinin (HA)-tagged GADD45 family members were cloned into pEBMulti-Hyg (Wako Pure Chemicals, Osaka, Japan). Cells constitutively expressing HA-GADD45 proteins were selected in medium supplemented with 100~300 µg/ml hygromycin B.
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