Superose 6 3.2 300

The harvested cells were thawed on ice and resuspended in solubilization buffer: 20 mM HEPES–NaOH pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 1% (w/v) protease inhibitor mix, and 2% (w/v) GDN. 5 ml of the solubilization buffer was added per gram of cells. The lysate was homogenized and incubated at 4 °C for 1 h. The lysate was centrifuged at 100,000 ×g for 1 h at 4 °C, and the cleared supernatant was incubated with pre-equilibrated streptavidin agarose beads (ThermoFisher, Pierce). After washing the beads three times with washing buffer (20 mM HEPES–NaOH pH 7.4, 150 mM NaCl, 0.02% (w/v) GDN, and 2 mM PMSF), the protein was eluted with 2.5 µM biotin in 20 mM HEPES–NaOH pH 7.4, 150 mM NaCl, and 0.02% (w/v) GDN. The eluate was concentrated with a 100-kDa cut-off concentrator (Amicon-Ultra 0.5 ml, Merck, Millipore). The PLCs were isolated by size exclusion chromatography (Superose 6 3.2/300, GE), and the peak fraction harvested for further analysis.