HEK293 cells were cultured in growth medium containing Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific) with 10% fetal bovine serum, 1% penicillin-streptomycin, and 5 mM glutamine. Cells seeded in 3.5- or 6-cm plates, after 1 day, were transfected with cDNA constructs using Polyfect (Qiagen) as per the manufacturer’s instructions. For the amino acid deprivation protocol, after 48 hours, the growth medium was replaced with either serum-free F12+ (D2906, Sigma-Aldrich) medium or F12− (D9785, Sigma-Aldrich; without l -Leucine) medium for 2 hours, and, wherever indicated, F12− containing cells were stimulated with leucine (3 mM) for 15 min, and cells were lysed to proceed with Western blotting. HEK293 cells were seeded in 12-well plates for 1 day and then were transfected with RasGRP1 shRNA along with control shRNA constructs using Polyfect (Qiagen), according to the manufacturer’s instructions. The growth medium was removed after 24 hours, and the cells were lysed to prepare for Western blotting. Jurkat cells were also cultured similarly.
D2906
Manufactured by Merck Group
Sourced in United States
D2906 is a piece of laboratory equipment used for scientific analysis and research. It serves as a core function in various experiments and testing procedures. The detailed specifications and intended use of this product are not available within the scope of this request.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using d2906
1
Cellular Amino Acid Deprivation Assay
2
BNIP3 Protein Expression in H9c2 Cells
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Cell culture was performed following the methods given in our previous publications [35 (link)]. BD1X Rat embryonal heart H9c2 cells (CRL-1446, ATCC, Manassas, VA, USA) were grown in DMEM (D2906, Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% FBS and 1.5 g/L sodium bicarbonate, and then incubated at 37 °C in a 5% CO2 incubator. Full lengths of the BNIP3 open reading frame were cloned and inserted into the BamHI site of pcDNA3-HA for BNIP3 protein expression. All plasmids were prepared using the AxyPrepTM Plasmid Maxiprep kit (Axygen, Inc., Union City, CA, USA) and were transfected into cells using GeneJuice® (Novagen, Merck, Darmstadt, Germany) transfection reagent according to the manufacturer’s guidelines. After 6 h, the cells were fed with fresh medium followed by drug treatment.
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