High affinity 3f10

Proteins from the extracellular matrix (ECM) of CHO cells (CT) and CHO cells stably transfected with an expression plasmid carrying a C-terminal hemaglutinin-tagged (HA) version of the human KAL1 cDNA (A1 [42 (link)]) were extracted as described previously [32 (link)]. Briefly, the cells were washed once with calcium/magnesium-free Hank’s Balanced Salt Solution (Gibco|Thermo Fisher Scientific, Waltham, MA, USA) and they were then incubated at 4 °C for 30 min in a gently rocking 10 cm diameter culture dish in 1 mL of 20 mM phosphate buffer (PB, pH 7.4), containing 350 mM NaCl and complete EDTA free protease inhibitor (Roche, Basiela, Switzerland). The ECM proteins released into the buffer were concentrated ten-fold with Amicon Ultra-4 Ultracel-30k (Millipore Corporation, Burlington, NJ, USA) and used in the chemotaxis experiments (ECM extracts). The presence of anosmin-1 was confirmed in Western blots probed with a peroxidase-conjugated rat monoclonal antibody against HA (High Affinity 3F10; Roche, Basiela, Switzerland). Equal amounts of protein from untransfected and transfected cells were used in all the in vitro experiments described below.

Western blotting was carried out as described [12] (link). PBP3, PBP1b and FtsN were revealed with respective polyclonal antibodies and FtsW was probed with monoclonal anti-HA-Peroxydase (HighAffinity (3F10) Roche).