Complete column

cDNA fragments (Gen9) encoding the Tezepelumab (AMG-157) lambda light chain, the IgG2 heavy chain54 , and the PCR-derived V_H-C_H1 heavy chain fragment (primers 5 and 6) (Supplementary Table 5) were cloned between the AgeI and KpnI sites of the pHLsec vector, in frame with the vector's signal peptide. At the C-terminus, the heavy chain and the V_H-C_H1 fragment carried a hexahistidine tag. The mAb and its Fab-fragment were produced by co-transfecting HEK293T cells with expression plasmids for the light chain and, heavy chain or V_H-C_H1-fragment in a 1:1 ratio. The mAb or Fab-fragment were purified from the conditioned medium by IMAC (Roche cOmplete column) and SEC (Superdex 200) using HBS pH 7.4 as running buffer. The Fab:TSLP complex was formed by incubating the Fab-fragment with a molar excess of refolded TSLP^Δ127–131 produced in E. coli as described above. The complex was then isolated from the molar excess of TSLP^Δ127–131 by SEC and concentrated to 10 mg ml⁻¹. The protein sample was then aliquoted and flash frozen in liquid nitrogen.

FLMyo6 SI (WT) or (SAHmimic) were used alone or mixed with partner GIPC1 (His-rTEV-GIPC1 255-end) or TOM1 (His-TOM1 207-492) in a ratio (1/1) (10 µM) and 1 µM of extra Calmodulin was added in a total volume of 20 µL. The input was incubated with 40 µL of Ni²⁺ beads from cOmplete column (Roche), which were previously equilibrated either in ADP.VO₄ Buffer (10 mM HEPES pH 7.5; 100 mM NaCl; 5 mM NaN₃; 1 mM MgCl₂; 0.1 mM TCEP; 1 mM NaADP; 1 mM Na₃VO₄; 0.1 mM EGTA; 4 mM imidazole) or ADP.VO₄-CaCl₂ Buffer (10 mM HEPES pH 7.5; 100 mM NaCl; 5 mM NaN₃, 1 mM MgCl₂, 0.1 mM TCEP, 1 mM NaADP; 1 mM Na₃VO₄; 4 mM imidazole; 1 mM CaCl₂) or NF Buffer (10 mM HEPES pH 7.5; 100 mM NaCl; 5 mM NaN₃; 1 mM MgCl₂; 0.1 mM TCEP; 0.1 mM EGTA; 4 mM imidazole). All steps were performed at 4 °C. Beads were washed by centrifugation after 1 h of gentle agitation. Bound proteins were eluted in 600 mM imidazole in the corresponding buffer.