HeLaM cells [56 (link)] and patient fibroblasts [9 (link)] were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) supplemented with 10% (v/v) foetal calf serum (Sigma), 2 mM L-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. The BAC-transgenic HeLa cell line expressing SPG15-GFP under its own promoter had to be regularly sorted by FACS due to loss of expression. HeLa cells expressing the reporter constructs composed of the cytoplasmic tail of CIMPR (CD8-CIMPR) or sortilin (CD8-sortilin) coupled to the transmembrane and lumenal domain of CD8 were kind gifts from Matthew Seaman [43 (link)].
For proteomics, HeLa cells were grown in SILAC medium supplemented with 10% (v/v) dialysed foetal calf serum (10,000 MW cutoff; Invitrogen), penicillin/streptomycin (Sigma), and either “Heavy” amino acids (L-arginine-13C615N4:HCl [50 mg/L] and L-lysine-13C615N2:2HCl [100 mg/L]; Cambridge Isotope Laboratories) or the equivalent “Light” amino acids. Cells were grown for at least 7 days to achieve metabolic labelling, and the average incorporation efficiency was approximately 95%, as determined by mass spectrometry.
For proteomics, HeLa cells were grown in SILAC medium supplemented with 10% (v/v) dialysed foetal calf serum (10,000 MW cutoff; Invitrogen), penicillin/streptomycin (Sigma), and either “Heavy” amino acids (L-arginine-13C615N4:HCl [50 mg/L] and L-lysine-13C615N2:2HCl [100 mg/L]; Cambridge Isotope Laboratories) or the equivalent “Light” amino acids. Cells were grown for at least 7 days to achieve metabolic labelling, and the average incorporation efficiency was approximately 95%, as determined by mass spectrometry.
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