Ng was cloned by PCR from a commercial rat brain cDNA (Clontech, Mountain View, CA, USA). GFP-Ng was made with pEGFP plasmid and re-cloned into pSinRep5 (Invitrogen, Grand Island, NY, USA) for virus preparation as described (Zhong et al., 2009 (link)). After 5–7 days in culture, GFP-Ng was delivered into the slices using the Sindbis virus expression system, which is a replication-deficient, low-toxicity and neuron-specific system (Malinow et al., 1999 ).
Psinrep5
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PSinRep5 is a laboratory instrument designed for the analysis of protein samples. Its core function is to perform quantitative measurements of protein expression levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Cy3 conjugated anti rabbit igg antibody, by Jackson ImmunoResearch (2 mentions)
Anti glua1 n, by Alomone (2 mentions)
Megascript sp6 kit, by Thermo Fisher Scientific (2 mentions)
Mmessage mmachine sp6 transcription kit, by Thermo Fisher Scientific (1 mentions)
Pgex 6p 1, by GE Healthcare (1 mentions)
β actin, by Merck Group (1 mentions)
Pdh26s, by Thermo Fisher Scientific (1 mentions)
Gene pulser 2, by Bio-Rad (1 mentions)
8 protocols using psinrep5
1
Recombinant Neuronal Protein Expression
2
Palmitoylated GFP Sindbis Virus Expression
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The DNA construct containing the sequences for GFP tagged with a membrane-targeting palmitoylation site was inserted into the PmaCI site of pSinRep5 (Invitrogen, Carlsbad, CA, USA) as fully described by Furuta et al. (2001 (link)). The recombinant Sindbis virus, which was produced with the pSinRep5 containing the construct, was replication deficient and designed so that infected cells would express palGFP under the control of a powerful sub-genomic promoter of the virus.
3
Sindbis Pseudovirion Production and Neuronal Infection
4
Sindbis Virus Expression of PTPN11 in Neurons
5
Sindbis Virus Expression of PTPN11 in Neurons
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The coding sequence of human PTPN11 with or without the D61G mutation was subcloned into a Sindbis viral expression vector (pSinRep5; Invitrogen) and GFP was inserted into the 3′ region of the coding sequence along with an additional subgenomic promoter for bicistronic expression. Sindbis viruses were produced according to the manufacturer’s protocol (Invitrogen) and directly added to the medium of cultured rat hippocampal neurons (DIV21). Twelve hours after infection, immunocytochemistry was performed with or without permeabilization by using anti-GluA1-N (#AGC-004, Alomone labs) antibody and Cy3–conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Lab). Images were acquired by using confocal microscope (Zeiss LSM 710) and analyzed by using ImageJ (ver. 1.42q).
6
Recombinant Expression Constructs
7
Sindbis Virus Production for FLII12Pglu-700μδ6 Sensor
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The glucose sensor FLII12Pglu-700μδ6 (Takanaga et al., 2008 (link)) was used in this study. The coding sequence of the sensor was subcloned into the viral vector pSinRep5 (Invitrogen). Sindbis virus were produced as previously described (Hepp et al., 2007 (link); Hu et al., 2011 (link)). Recombinant pSinRep5 and helper plasmid pDH26S were transcribed in vitro into capped RNA using the Megascript SP6 kit (Ambion). Baby hamster kidney-21 (ATGC #CCL-10) cells were electroporated with sensor-containing RNA and helper RNA (2 × 107 cells, 950 μF, 230 V) and incubated for 24 h at 37°C in 5% CO2 in Dulbecco’s modified Eagle Medium supplemented with 5% fetal calf serum before collecting cell supernatant containing the viruses. The virus titer (108 infectious particles/ml) was determined after counting fluorescent baby hamster kidney cells infected using serial dilution of the stock virus.
8
Sindbis Virus Production Protocol
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The GA sequence (Genbank accession number: EF212028) has been described elsewhere (Drobac et al. 2010) and was similar to that of the G5A fusion protein reported previously (Baubet et al, 2000) . GA was inserted into the plasmid pSinRep5 (Invitrogen, Carlsbad, CA, USA) between SphI and ApaI sites. The resulting plasmid and the helper plasmid pDH26S (Invitrogen) were linearized with PacI and XhoI, respectively; and in vitro transcribed into capped RNA using the Megascript SP6 kit (Ambion, Huntingdon, UK).
Sindbis virus production was performed as described (Drobac et al. 2010) . BHK-21 cells (ATCC # CCL-10) were electroporated (20.10 6 cells/ml, 950 μF, 230 V) with recombinant and helper viral RNAs using the Gene Pulser II electroporation system (Bio-Rad Laboratories, Hercules, CA, USA) and grown 24 h at 37°C, 5% CO2 in DMEM containing 5% fetal calf serum before collecting the cell supernatant containing viral particles. The recombinant virus titer (1.8x10 8 ip/ml) was determined after counting fluorescent BHK cells infected using serial dilution of the virus stock.
Sindbis virus production was performed as described (Drobac et al. 2010) . BHK-21 cells (ATCC # CCL-10) were electroporated (20.10 6 cells/ml, 950 μF, 230 V) with recombinant and helper viral RNAs using the Gene Pulser II electroporation system (Bio-Rad Laboratories, Hercules, CA, USA) and grown 24 h at 37°C, 5% CO2 in DMEM containing 5% fetal calf serum before collecting the cell supernatant containing viral particles. The recombinant virus titer (1.8x10 8 ip/ml) was determined after counting fluorescent BHK cells infected using serial dilution of the virus stock.
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