pCEP-Pu

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	Human embryonic kidney (HEK) 293-EBNA cells (Invitrogen) grown in Dulbecco's Modified Eagle Medium (Invitrogen) according to our previously published procedure [14] were grown to three-fourth confluency and transfected for 24 h with Lipofectamine 2000 (Invitrogen) using different concentrations of pCEP-Pu plasmid DNA containing the gpTNF-α cDNA. The transfection medium was then replaced with CD-293 medium without serum containing puromycin (0.05 μg/mL) and the cells were incubated at 37°C for 48 h before collecting the supernatant fluid.

Protocol tips

Human embryonic kidney (HEK) 293-EBNA cells (Invitrogen) grown in Dulbecco's Modified Eagle Medium (Invitrogen) according to our previously published procedure [14] were grown to three-fourth confluency and transfected for 24 h with Lipofectamine 2000 (Invitrogen) using different concentrations of pCEP-Pu plasmid DNA containing the gpTNF-α cDNA. The transfection medium was then replaced with CD-293 medium without serum containing puromycin (0.05 μg/mL) and the cells were incubated at 37°C for 48 h before collecting the supernatant fluid.

Publication protocol

Human embryonic kidney (HEK) 293-EBNA cells (Invitrogen) grown in Dulbecco's Modified Eagle Medium (Invitrogen) according to our previously published procedure [14] were grown to three-fourth confluency and transfected for 24 h with Lipofectamine 2000 (Invitrogen) using different concentrations of pCEP-Pu plasmid DNA containing the gpTNF-α cDNA. The transfection medium was then replaced with CD-293 medium without serum containing puromycin (0.05 μg/mL) and the cells were incubated at 37°C for 48 h before collecting the supernatant fluid. Purification of cell lysates containing the putative rgpTNF-α was performed in a similar manner to that described above for prokaryotic expressed rgpTNF-α.

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