At the end, resin was briefly centrifuged in order to recover the supernatant (unbound fraction) and successive washes with equilibration buffer were performed in order to remove all unbound proteins.
To test the strength of anchoring of the diverse cells to plates, a protocol was performed in which 4 consecutive washes with isotonic buffer were performed, followed by the use of a colorimetric protocol for counting the cells that remained in the plate (based on the vital dye neutral red).
Buffer titrations were performed with a solution of 10M NaOH in water to adjust pH.
Three buffer exchanges were performed with a spin filter device of 5,000 Da molecular weight cut-off to reduce salt concentration before storing at −80°C until further use.
Beads from immunoprecipitations were washed three times in lysis buffer and once in kinase buffer, and kinase assays were performed with recombinant GST-S6 as substrate (1 μg per assay).
After 48 hours, cells were harvested in 100 μl reporter lysis buffer and luciferase assays were performed with 20 μl of lysates using the Luciferase assay system (Promega, Madison, WI, USA).
DNAs were resuspended in TE buffer, and PCR analyses were performed with the following primers: p21 (forward: 5′-GTAAATCCTTGCCTGCCAGAGTG-3′ reverse: 5′-GCTGCCCAGCGCCGAGCCAG-3′) and TOP2A (forward: 5′-CGTCAGAACAGAGGACAGTTTTT-3′ reverse 5′-TGGAAGAGATGGGCTTTGG-3′).
For immobilization of ethanolamine as a reference column, 10 × 20-μL injections of ethanolamine (6.15 mg/mL) and sodium cyanoborohydride (9 mg/mL) dissolved in 0.1 M sodium phosphate buffer at pH 7.0 were performed with a flow rate of 20 μL/min for 1 min.
Porphysomes solutions were diluted in buffered saline, three measurements were performed with 15 runs each, and the results were averaged.
