Generation of a LOX-1 cDNA expression plasmid
Total RNA was extracted from HSCs using Trizol reagent and reversely transcribed into cDNA, which was used as a template for PCR amplification of LOX-1 cDNA. The PCR primers were designed according to GenBank accession number: NM_138648, and use the following primers: (F) 5′-CCC AAG CTT ATG ACT TTT GAT GAC AAG ATG AAG C-3′; (R) 5′-GG GGT ACC CC CTA AAT TTG CAA ATG ATTT GTC TTC-3′. The primers were tailed with HindIII site (the forward primer) and KpnI site (the reverse primer). The PCR product was subcloned into pCDNA3.1/Zeo+ expression vector (Invitrogen Corp. Carlsbad, CA) at HindIII/KpnI sites. The subcloned LOX-1 cDNA was verified by DNA sequencing.