HEK293T cells were obtained from DSMZ and cultured in DMEM (Life Technologies) with 10% fetal bovine serum (Life Technologies), 1% penicillin/streptomycin (Life Technologies), and 1% l -glutamine (Life Technologies). All cells were maintained at 37 °C in a humidified incubator containing 5% CO2 and routinely tested for mycoplasma infection. For SILAC labeling, cells were maintained in media containing either l -arginine and l -lysine (light SILAC label), l -arginine (13C6) and l -lysine (2H4) (medium SILAC label), or l -arginine (13C6-15N4) and l -lysine (13C6-15N2) (heavy SILAC label) (Cambridge Isotope Laboratories).
L arginine
Manufactured by Cambridge Isotopes
Sourced in United States
L-arginine is a naturally occurring amino acid that plays a crucial role in various physiological processes. It is an essential component for the synthesis of proteins and serves as a precursor for the production of nitric oxide, which is involved in regulating blood flow and other important functions. L-arginine is commonly used in laboratory settings for research and analytical purposes.
Automatically generated - may contain errors
Lab products found in correlation
L lysine, by Cambridge Isotopes (6 mentions)
L arginine 13c615n4 and l lysine 13c6 15n2, by Cambridge Isotopes (2 mentions)
Silac mem medium, by Thermo Fisher Scientific (2 mentions)
Light l proline, by Merck Group (2 mentions)
Gibco cell dissociation buffer, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
13c615n4 l arginine, by Cambridge Isotopes (2 mentions)
13c6 l arginine, by Cambridge Isotopes (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by American Type Culture Collection (1 mentions)
Oci aml3 cells, by Leibniz Institute DSMZ (1 mentions)
Dmem medium, by PAN Biotech (1 mentions)
L arginine and l lysine, by Cambridge Isotopes (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
L arginine 13c6 and l lysine 2h4, by Cambridge Isotopes (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
11 protocols using l arginine
1
SILAC Labeling of HEK293T Cells
2
SILAC Labeling of Cell Lines
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U2OS, HCT116, and HEK293T cells were obtained from ATCC and cultured in D-MEM medium (U2OS and HEK293T) or RPMI 1640 (HCT116) supplemented with 10% fetal bovine serum, l -glutamine, penicillin, and streptomycin. OCI-AML3 cells were purchased from DSMZ GmbH and cultured in a D-MEM medium (PAN-Biotech) containing 20% FBS, l -glutamine, penicillin, and streptomycin. Cells were routinely tested for mycoplasma infection with a PCR-based method. For SILAC labeling, cells were cultured in media containing either l -arginine and l -lysine, l -arginine [13C6], and l -lysine [2H4] or l -arginine [13C615N4] and l -lysine [13C6-15N2] (Cambridge Isotope Laboratories)79 . All cells were cultured at 37 °C in a humidified incubator containing 5% CO2.
3
SILAC Metabolic Labeling Protocol
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Cells were grown in SILAC MEM medium (Thermo Fisher Scientific) lacking lysine and arginine, supplemented with dialysed 10% FBS (Sigma-Aldrich), 200 mg/l light L-Proline (Sigma-Aldrich) and either heavy L-Lysine (13C615N2; 146 mg/l) and L-Arginine (13C615N4; 84 mg/l) (Cambridge Isotope Laboratories) or their light equivalents (Sigma-Aldrich). Cells were passaged with non-enzymatic Gibco Cell Dissociation Buffer (Thermo Fisher Scientific). >95% heavy amino acid incorporation rate was validated by mass-spectrometry.
4
SILAC Labeling and Transfection Protocol
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U2OS, HEK293T, RPE-1, and HaCaT cells were obtained from ATCC or DSMZ and cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, l -glutamine, penicillin, and streptomycin. Cells were routinely tested for mycoplasma infection with a PCR-based method. For SILAC labeling, cells were cultured in media containing either l -arginine and l -lysine, l -arginine [13C6] and l -lysine [2H4] or l -arginine [13C615N4] and l -lysine [13C6-15N2] (Cambridge Isotope Laboratories)50 (link). All cells were cultured at 37 °C in a humidified incubator containing 5% CO2. Cells were transfected with siRNAs using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. The complete list of siRNA sequences and oligos used in this study can be found in Supplementary Tables 1 and 2 , respectively.
5
SILAC Labeling of U2OS and HEK293T Cells
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U2OS and HEK293T cells were obtained from ATCC and cultured in d -MEM medium supplemented with 10% fetal bovine serum, l -glutamine, penicillin, and streptomycin. Cells were routinely tested for mycoplasma infection with a PCR-based method. For SILAC labeling, cells were cultured in media containing either l -arginine and l -lysine, l -arginine [13C6] and l -lysine [2H4] or l -arginine [13C615N4] and l -lysine [13C6-15N2] (Cambridge Isotope Laboratories) as described previously (62 (link)). All cells were cultured at 37°C in a humidified incubator containing 5% CO2.
6
SILAC Metabolic Labeling Protocol
7
Metabolic Labeling for Proteomic Analysis
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Cells were incubated
for 30 min in custom-made RPMI (Gibco) depleted of arginine, lysine,
and methionine and supplemented with 10% dialyzed FBS (Gibco), glutamine,
penicillin, and streptomycin. Next, the same medium was supplemented
with 0.1 mMl -γ-azidohomoalanine (AHA) (Bachem) or l -methionine as control. Additionally, for p-SILAC experiments,
the following reagents were used per labeling condition: intermediate
(200 μg/mL [13C6]l -arginine,
40 μg/mL [4,4,5,5-D4]l -lysine (Cambridge
Isotope Laboratories)) or heavy (200 μg/mL [13C6, 15N4]l -arginine, 40 μg/mL
[13C6,15N2]l -lysine (Cambridge Isotope Laboratories)). For label-free experiments,
200 μg/mLl -arginine and 40 μg/mL l -lysine
(Cambridge Isotope Laboratories) were used.
for 30 min in custom-made RPMI (Gibco) depleted of arginine, lysine,
and methionine and supplemented with 10% dialyzed FBS (Gibco), glutamine,
penicillin, and streptomycin. Next, the same medium was supplemented
with 0.1 mM
the following reagents were used per labeling condition: intermediate
(200 μg/mL [13C6]
40 μg/mL [4,4,5,5-D4]
Isotope Laboratories)) or heavy (200 μg/mL [13C6, 15N4]
[13C6,15N2]
200 μg/mL
(Cambridge Isotope Laboratories) were used.
8
Comprehensive Lipid and Metabolite Analysis
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PC(12:0/13:0), PE(12:0/13:0), SM(d18:1/12:0) and Cer(d18:1/12:0) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Stable isotope labeled references were purchased from Cambridge Isotope Laboratories (Andover, MA, USA), including: Glycine (2-13C, 15N), L-Alanine (2,3,3,3-D4), L-Valine (D8), L-Leucine (5,5,5-D3), L-Methionine (methyl-D3), L-Phenylalanine (ring-13C6), L-Tyrosine (ring-13C6), L-Aspartic acid (2,3,3-D3), DL-Glutamic acid (2,4,4-D3), L-Ornithine: HCl (5,5-D2), L-Citrulline (5,5-D2) and L-Arginine (5-13C, 4,4,5,5-D4). Deuterium-labeled carnitine and acylcarnitines (NSK-B set) were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA), including 2H3-Acetylcarnitine (C2), 2H3-Propionylcarnitine (C3), 2H3-Butyrylcarnitine (C4), 2H9-Isovalerylcarnitine (C5), 2H3-Octanoylcarnitine (C8), 2H9-Myristoylcarnitine (C14), and 2H3-Palmitoylcarnitine (C16). HPLC-grade ammonium acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA), and HPLC-grade formic acid and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All solutions were prepared by LC-MS ultra-pure water.
9
SILAC-Based Proteomic Analysis of Selinexor
10
SILAC-based Immunoprecipitation and Quantitative Proteomics
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Cells were cultured in DMEM medium without lysine or arginine, and supplemented with dialyzed serum for two weeks prior to the experiment. Isotopes of L-lysine and L-Arginine (Cambridge Isotope Laboratories) were added to the medium to label the following three SILAC-states: Light (K0R0), Medium (K4R6), and Heavy (K8R10). Immunoprecipitations were performed for each state separately, as described above. As published previously [101 (link)], bound proteins of all three states were mixed after immunoprecipitation and digested “on bead”. Data were analyzed using the Andromeda Search Engine within the MaxQuant software package, version 1.5.3.8 [102 (link),103 (link)].
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