Rabbit polyclonal antibodies against sortilin (Abcam, ab16640, 1:1000), rabbit monoclonal antibody against chlamydial protease/chaperone protein, CtHtrA (Huston et al., 2008 (link)) and mouse monoclonal antibodies against beta-actin (Merck Millipore, MAB1501, 1:1000) were used. Secondary antibodies were purchased from Molecular Probes (Life Technologies) and Li-Cor Bioscience. mCherry-Rab25 was obtained by performing restriction digest using restriction enzymes BamHI and EcoRI on GFP-Rab25 (Casanova et al., 1999 (link)) to obtain the open reading frame of Rab25 and subcloned into mCherry-C1 following standard protocols. Stably expressing mCherry-Rab25 HeLa cells were generated by transient transfection of mCherry-Rab25 into HeLa cells using Lipofectamine 2000 as per manufacturer's instruction (Invitrogen). Transfected cells were selected using 400 µg/ml of G418 over a period of 14 days to generate stable cell lines expressing mCherry-Rab25. pGIPZ small hairpin RNA (shRNA) plasmids (Thermo Scientific) used included non-silencing control shRNA: RHS4346; human sortilin shRNAs (shRNA #1: V2LHS_31931, shRNA #2: V2LHS_31928, shRNA #3: V3LHS_359770, shRNA #4: V3LHS_359767, shRNA #5: V3LHS_359771) were supplied by the Institute for Molecular Bioscience Life Science Automation (LISA) Facility.
Rhs4346
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RHS4346 is a laboratory instrument designed for performing various tasks in a research or analytical setting. It is a versatile piece of equipment that can be utilized for a range of applications. The core function of the RHS4346 is to facilitate the processing and analysis of samples, though the specific details of its intended use are not provided.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
Ab16640, by Abcam (1 mentions)
Pgipz small hairpin rna shrna plasmids, by Thermo Fisher Scientific (1 mentions)
Mab1501, by Merck Group (1 mentions)
Lenti x 293t cells, by Takara Bio (1 mentions)
Slhv033rs, by Merck Group (1 mentions)
Rhs4080, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Ab19862, by Abcam (1 mentions)
Odyssey blocking buffer obb, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Gipz lentiviral shrnamir vectors, by Thermo Fisher Scientific (1 mentions)
Sb415286, by Selleck Chemicals (1 mentions)
Etomoxir, by MedChemExpress (1 mentions)
Oil red o, by Merck Group (1 mentions)
Pmd2 g envelope, by Addgene (1 mentions)
Pspax2, by Addgene (1 mentions)
18 protocols using rhs4346
1
Generating mCherry-Rab25 HeLa Cell Line
2
Lentiviral Transduction of Fgf11 Knockdown
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Lenti-X 293 T cells (Clontech, 632180) were seeded on 100 mm dishes and transfected with psPAX2 packaging plasmid (6 μg), pMD2.G envelope plasmid (2 μg), and GPIZ constructs (8 μg, green fluorescent protein (GFP), carrying either control (Dharmacon, RHS4346) or Fgf11-targeting shRNA using TurboFect (Thermo Scientific, R0531) following the manufacturer’s instructions. To prevent off-target effects, 2 different shRNA plasmids targeting both A and B isoforms of Fgf11 (Dharmacon, VGM5520-200406248 and VGM5520-200407071) were selected after testing 6 different shRNA plasmids for mouse Fgf11. Culture medium containing lentiviruses was harvested and filtered through 0.45 μm syringe filters (Millipore, SLHV033RS) as previously described [29 (link)]. To obtain concentrated viruses, 4 successive rounds of ultracentrifugation were carried out in the same ultra-clear centrifuge tubes (Beckman, 344058) at 43,000 × g for 120 min at 4 °C [30 (link)]. After final centrifugation, pellets were gently resuspended in saline. The titers of lentiviral stocks were determined by flow cytometry [31 (link)]; that of control virus was 6.76 × 1010 and that of shFgf11-expressing lentivirus was 6.62 × 1010 IU/ml. The concentrated viruses were aliquoted and stored at − 80 °C.
3
Transient PRDM2 Knockdown in PRDM2insG and HCT116 Cells
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The pGIPZ lentiviral shRNA vector targeting PRDM2 (RHS4430-200269018) as well as a non-silencing pGIPZ control vector (RHS4346), both from Thermo Scientific, were used to transiently transfect PRDM2insG clone 1 and HCT116 cells. Cells were seeded in 6–well plates and transfection was performed with Lipofectamine 2000 (Life Technologies) using 5 μg of recombinant construct. Cells were harvested 48 h post transfection and RNA was extracted and cDNA synthesized as previously described. RT-qPCR for VCAN, EMP3, CPA4, VIM, VSNL1, SUSD2, KRT23, MMP14 and TMTC1 was carried out as mentioned above.
4
Plexin-B2 Knockdown Using GIPZ Lentivirus
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Stable knockdown of Plexin-B2 was achieved with GIPZ lentiviral vectors expressing shRNA against Plexin-B2, along with GFP and Puromycin resistance markers (Thermo Scientific, RHS4430-100986735 (shRNA PB2#1) and RHS4430-100989559 (shRNA PB2#2)). A GIPZ vector with non-targeting shRNA served as control (Thermo Scientific RHS4346). Lentiviral particles were produced with 293T cells co-transfected with GIPZ plasmid, envelope plasmid pMD2.G, and packaging plasmid psPAX2 (Addgene plasmids 12259 and 12260; deposited by Didier Trono, EPFL Lausanne). Stable GIPZ sublines were established by Puromycin (1 μg/ml) selection.
5
Lentiviral Knockdown of Pdxdc1 in Mice
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PND63 male C57BL/6J mice were housed 1 week at The Centre for Phenogenomics (TCP; Toronto, ON, Canada), 5 mice per cage with 12 h light-dark cycle (0700–1900), 50–60% humidity, at 21±1 °C, with ad libitum food and water. Mice were pre-tested for PPI, then injected with virus. Experiments were conducted as per Figure 1a . GIPZ constructs for mouse Pdxdc1 shRNAmir V2LMM_63165, V3LMM_453828, non-silencing GIPZ shRNAmir negative control RHS4346 (Thermo Fisher Scientific, Waltham, MA, USA) were prepared as per kit directions and sent to SIDNET (The Hospital for Sick Kids, Toronto, ON, Canada) for packaging with Open Biosystems TransLenti Viral GIPZ Packaging System (Thermo Fisher Scientific). Production, concentration and titering were as per the GIPZ lentiviral product manual. Titers: 1.64 × 109 TU ml−1 (V2LMM_631165), 1.02 × 109 TU ml−1 (V3LMM_453828), and 2.1 × 108 TU ml−1 (RHS4346).
6
Overexpression of CHD7 and PAX6 in Cells
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Human full-length CHD7 cDNA (pF1KE9669 Flexi ORF Clone, PROMEGA, www. promega.jp) was subcloned into the pF5K CMV-neo Flexi vector vector (Promega), and a pMX-Pax6 plasmid (mouse) was obtained from Addgene (Cambridge, MA, www.addgene.org ). Human PAX6 expression vector (pCS-PAX6, BC011953) was purchased from Transomic (Huntsville, AL, www.transomic.com ). Recombinant lentiviruses were produced by the shCHD7 lentivirus vector (EHS4430-98514866, EHS4430-98714285, Open Biosystems. dharmacon.gelifesciences. com) or control shRNA vector (RHS4346, Open Biosystems). Retrovirus and lentivirus production including shMIF were performed as described previously [7 (link), 8 (link)].
7
Knockdown of RBM14 in GBM Spheres
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8
Generating murine IRF9 knockdown cell lines
9
Western Blot Analysis of SCD1 Protein
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Cells were lysed with SDS lysis buffer (1% SDS, 10 mM EDTA, 1x EDTA-free protease inhibitors) and protein concentration was determined by the bicinchoninic acid assay method (BCA, Pierce) according to the manufacturer protocol. Samples were boiled for 5 min after the addition of 6 ×x Laemmli buffer (9% SDS and 60% glycerol, 375 mM Tris-HCl pH 6.8, 0.015% Bromophenol blue, 12% β-mercaptoethanol). Proteins were separated for 2 hr on a 12% SDS-polyacrylamide gel using a Bio-Rad mini-PROTEAN electrophoresis apparatus, transferred to low-fluorescence PVDF membranes (Millipore) for 2 hr at 4°C at a constant 70 V in 25 mM Tris, 150 mM glycine, and 10% (vol/vol) methanol transfer buffer, blocked with Odyssey Blocking Buffer (OBB, LI-COR Biosciences) for 1 hr at room temperature, and probed with 1:1000 dilution of mouse monoclonal anti-SCD antibody (ab19862; Abcam) in OBB overnight at 4°C. After washing with PBS-T, membranes were incubated with fluorescent goat anti-mouse secondary antibodies (926-32210; LI-COR Biosciences) at 1:15000 in OBB for 1 hr at room temperature. Signal was detected using an Odyssey Infrared Imaging System (LI-COR Biosciences). To ensure antibody specificity, shRNA knockdown of SCD1 was performed with two independent shRNA constructs (V3LHS_305870 and V3LHS_305872; Open Biosystems) and compared with non-silencing shRNA construct (RHS4346; Open Biosystems).
10
Cell Culture and Genetically Modified Cell Lines
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