| growth | SILAC I (UR255): Cells were cultured in DMEM for SILAC medium (Thermo Fisher Scientific, #89985) containing light- (12C6,14N4–Arg, 12C6,14N2–Lys) (Sigma, #L5751, #A6969) or medium-labeled (13C6-Arg, 4D-Lys) (Cambridge Isotope Laboratories, #CLM-2265-H, #DLM-2640-0) amino acids. Four days after the start of amino acids labeling, cells were transfected with siRNA targeting endogenous TPR (Thermo Fisher Scientific, Silencer Select siRNA ID #s14353) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, #13778075). Five days after the start of amino acids labeling, cells growing in the medium with light-labeled amino acids were transfected with the vector expressing a siRNA-resistant version of TPR (pcDNA4/TO-TPR-GFP-siRNA-res) using Lipofectamine 2000 (Thermo Fisher Scientific, # 11668019). In parallel, the cells growing in DMEM medium containing medium-labeled amino acids were transfected with empty vector (pcDNA4/TO-GFP), serving as a negative control for the immunoprecipitation. At day seven, cells were harvested, and TPR-GFP and GFP were immunoprecipitated using the GFP-Trap Agarose beads (Chromotek, # gta-20).
SILAC II (UR302): Cells were cultured in DMEM for SILAC medium (Thermo Fisher Scientific, #88364) containing light- (12C6,14N4–Arg, 12C6,14N2–Lys) (Sigma, #L5751, #A6969) or heavy-labeled (13C6,15N4–Arg, 13C6,15N2–Lys) (Cambridge Isotope Laboratories, #CNLM-539-H, #CNLM-291-H) amino acids. At day five after the start of amino acids labeling, the cells growing in a medium with light-labeled amino acids were transfected with siRNA Control (Thermo Fisher Scientific, Silencer Select Negative Control #1). In parallel, cells growing in a medium containing heavy-labeled amino acids were transfected with siRNA targeting TPR (Thermo Fisher Scientific, Silencer Select siRNA ID #s14353), serving as a negative control for the immunoprecipitation. At day eight, cells were harvested, and endogenous TPR was immunoprecipitated using anti-TPR antibody (Atlas Antibodies, #HPA019661) coupled with the rec-Protein G-Sepharose 4B Conjugate beads (Thermo Fisher Scientific, # 10-1243).
SILAC III (UR304 ): Cells were growing in the DMEM for SILAC medium (Thermo Fisher Scientific, #88364) containing light- (12C6,14N4–Arg, 12C6,14N2–Lys) (Sigma, #L5751, #A6969) or heavy-labeled (13C6,15N4–Arg, 13C6,15N2–Lys) (Cambridge Isotope Laboratories, #CNLM-539-H, #CNLM-291-H) amino acids. Ten days after the start of the labeling with the amino acids, endogenous TPR was immunoprecipitated using anti-TPR antibody (Atlas Antibodies, #HPA019661) coupled with the rec-Protein G-Sepharose 4B Conjugate beads (Thermo Fisher Scientific, # 10-1243) from the cell growing in DMEM medium containing light-labeled amino acids. In parallel, cells growing in DMEM medium with the heavy-labeled amino acids were subjected to control immunoprecipitation using IgG antibody (Santa Cruz Biotechnology, #sc-2027), serving as a negative control for the immunoprecipitation. |
