Cells were harvested, washed and lysed by passage through a French press pressure cell as described previously [ 26].
Subsequently, they were ruptured by 3-fold passages through a French Press cell at 18,000 p.s.i.i
Cells were lysed by three passes through a pre-cooled small French Press cell (Thermo IEC, Waltham, MA, USA) at 12,500 psi.
As we have previously reported (Vekrellis et al., in press), cell death occurred in this setting, unlike the situation in the cycling cells.
After 24 h, cells were harvested by centrifugation, resuspended in 50 mM sodium phosphate buffer, pH 8.0, containing 300 mM NaCl and disrupted by several passages through a French Press cell.
After overnight culture at 16°C, cells were harvested and disrupted with a French Press cell system, in a lysis buffer (pH 8) containing 100 mM Tris-HCl, 150 mM NaCl, 14 mM MgCl2 and 1 mM ATP. DNase and a protease inhibitor cocktail (Sigma-Aldrich) were added.
Inclusion bodies were obtained by French press cell-disruption and solubilised according to Kojima's method [ 36].
As previously reported, these mAbs possess mid (ABT-207) and high (h5F9-AM8) binding affinity towards repulsive guidance molecule A (RGMa) (Demicheva et al. in press Cell Reports 2015) and RGMc.
Moreover, MiMICS in coding introns have recently been used to create gene specific GAL4 lines that permit one to reveal where genes are expressed when endogenous protein levels are low (Diao et al., 2014, in press Cell Reports; Gnerer et al., 2015, in press Nucleic Acids Research).
Cell paste (∼7 g) was washed twice with 50 mL of buffer A and then suspended in 28 mL (4 mL/g of cell paste) of buffer A containing protease inhibitors (Roche) and subjected to two passes through a French press cell (14000 psi).
