Generation and Characterization of Boc Deletion Mutants

pCIG-mCdon-HA (pCIG-mCdon), pCIG-rCdon-FnIIIΔ12-HA (CdonΔFnIII(1-2)) and pCIG-mCdon-FnIIIΔ3-HA (CdonΔFnIII(3)) vectors were kindly provided by Dr B. Allen17 (link). pEGFP-mBoc expression vector (Boc-EGFP) was kindly provided by Dr Ami Okada65 (link). Versions of Boc lacking FnIII-2 or the FnIII-3 were generated by PCR amplification with a PfuUltra DNA polymerase (Agilent) from the pEGFP-mBoc plasmid using the following primers: Boc-GFPΔFnIII-2 fwd: 5′-GTGTCAGGCTACAGTGGC-3′, rev: 5′-ATGGTCAGGCTGGCTGCT-3′; Boc-GFPΔFnIII-3 fwd: 5′-TTTTCTGGTCAGCCTGGA-3′, rev: 5′-CCTCTCATATACACGGCC-3′. The amplified products were ligated (Ligase, Roche) overnight at 16 °C, digested with DpnI enzyme to remove the non-amplified DNA and then transformed.