To generate the BioID2-HA plasmid, BioID2 was PCR-amplified from the MCS-BioID2-HA plasmid (Addgene #74224) using primers designed to introduce an ATG start codon immediately downstream of the BamHI restriction site of the MCS. The PCR product was cut by BamHI and HindIII and cloned into the MCS-BioID2-HA plasmid (Addgene #74224). To generate the MICU1-BioID2-HA, MICU1 was PCR amplified from the hMICU1-Myc-DDK plasmid using primers to introduce a 5’ AgeI and a 3’ BamHI restriction site. The PCR product was cut by AgeI and BamHI and cloned into the MCS-BioID2-HA plasmid (Addgene #74224). The MICU1-HA plasmid was generated by cleaving the MICU1 fragment from the MICU1-FLAG plasmid (Origene # MR207652) using the SgfI-MluI restriction sites and inserted into the same sites in pCMV6-AC-HA vector (Origene # PS100004). Flag-tagged Mouse MICU1 (NM_144822) mutants; EMREmut KKKKR101-105QQQQQ; EF1mut D233A, E244A; EF2mut D423A, E434A; EF1/2mut D233A, E244A, D423A, E434A; MCUmut K440A, R442A, R445A; mtCUmut KKKKR101-105QQQQQ, K440A, R442A, R445A; Dimermut C465A; Truncation 1 (T1): Δ60 – 134; Truncation 2 (T2): Δ135 – 220; Truncation 3 (T3): Δ221 – 314; Truncation 4 (T4): Δ315 – 399; Truncation 5 (T5): Δ400 – 477 were custom cloned by Vector Builder Inc. Plasmids were confirmed by restriction digestion and DNA sequencing. Specific details of plasmid sources are provided in table S1 .
Pcmv6 ac ha vector
Manufactured by OriGene
Sourced in United States
The PCMV6-AC-HA vector is a plasmid used for the expression of proteins with a hemagglutinin (HA) tag in mammalian cells. The vector contains the cytomegalovirus (CMV) promoter for high-level expression of the target protein, as well as the HA tag sequence for detection and purification purposes.
Automatically generated - may contain errors
Lab products found in correlation
Mcs bioid2 ha, by Addgene (1 mentions)
Mcs bioid2 ha plasmid, by Addgene (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Hepg2 cell, by American Type Culture Collection (1 mentions)
Pflag cmv 2 expression plasmid, by Merck Group (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
3 protocols using pcmv6 ac ha vector
1
Engineered MICU1 Plasmid Construction
2
Generating Stable Cell Lines Expressing Proteins
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MYC-tagged SPHK1 was created in the pCMV6-AN-MYC (PS100012, Origene) vector by standard subcloning. CDH1 was constructed in the pCMV6 vector (PS100001, Origene) by standard subcloning. HA-tagged BECN1 was constructed in the pCMV6-AC-HA vector (PS100004, Origene) by standard subcloning. Flag-tagged TRAF2 and its mutant (ΔRING) were cloned into the pFLAG-CMV-2 expression plasmid (E7033, Sigma).
Human liver carcinoma HepG2 cells were purchased from American Type Culture Collection (ATCC, HB-8065) and maintained according to their instructions. Transient transfection was performed using Lipofectamine 2000 (Invitrogen, 11668019) or Lipofectamine RNAiMax (Invitrogen, 13778150) according to the manufacturer's instructions. To generate HepG2 cells stably expressing SPHK1, the pCMV6-AN-MYC and the MYC-tagged SPHK1 plasmids were both transfected into HepG2 cells. After 24 h of transfection, stable transfectants were selected using neomycin, and a single clone was amplified using a dilution-cloning technique.
Human liver carcinoma HepG2 cells were purchased from American Type Culture Collection (ATCC, HB-8065) and maintained according to their instructions. Transient transfection was performed using Lipofectamine 2000 (Invitrogen, 11668019) or Lipofectamine RNAiMax (Invitrogen, 13778150) according to the manufacturer's instructions. To generate HepG2 cells stably expressing SPHK1, the pCMV6-AN-MYC and the MYC-tagged SPHK1 plasmids were both transfected into HepG2 cells. After 24 h of transfection, stable transfectants were selected using neomycin, and a single clone was amplified using a dilution-cloning technique.
3
Overexpression of GRβ in Dysplastic HBMECs
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To simulate the increase in GRβ expression observed in the dysplastic tissue compared to non-dysplastic, HBMECs were transfected with HA-tagged GRβ DNA (1.075 µg/µL). The custom HA-tagged GRβ plasmid was obtained from OriGene Technologies utilizing the open reading frame (ORF) from cat. RC220377 (OriGene Technologies, Rockville, MD, USA) cloned in a pCMV6-AC-HA vector (OriGene Technologies, cat. PS100004). To achieve this transfection, 5 µg of HA-GRβ DNA was mixed in serum-free Dulbecco’s modified eagle medium (DMEM/F12) and was later combined with a 30 µg mixture of lipofectamine (Thermo Fisher Scientific, cat. 18324-012) in serum-free DMEM to form the DNA+lipofectamine complex. This mixture was set aside for 25 min at room temperature. Once formed, this DNA+lipofectamine complex was combined with additional serum-free DMEM and added to the 100 mm Petri dish of 70% confluent HBMECs and left to incubate for 5 h at 37 °C. After the incubation was complete, the serum-free media containing the DNA+lipofectamine complex was aspirated, the plate was washed with 0.1 M phosphate-buffered saline (PBS), and the PBS was replaced with normal HBMEC media (Cell Systems, cat. 4Z0-500) until the following day when subsequent drug treatment experiments were performed. These transfected cells will be denoted throughout as “HBMEC+HA-GRβ”.
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