CLIC1 and CLIC4 Knockdown in HUVEC

Human CLIC1 and CLIC4 shRNA-containing constructs were purchased from Sigma-Millipore and screened for significant CLIC1 and CLIC4 knockdown in HUVEC by immunoblotting as described below. Screenings of the shRNAs for both proteins were reported previously (20 (link), 21 (link)). Human CLIC4 shRNA targets the 5’-UTR regions with targeting sequence 5′-GCCGTAATGTTGAACAGAATT-3′, and human CLIC1 shRNA targets the coding region with targeting sequences 5’-CCTGTTGCCAAAGTTACACAT-3’ (Sigma). A lentiviral vector expressing scrambled shRNA was used as a control (Sigma-Millipore). Recombinant CLIC1 and CLIC4 DNA inserts were placed into the lentiviral pCCL vector. Wild type CLIC1 and CLIC4 were generated from cDNA of HUVECs. CLIC1 and CLIC4 variants were created by insertion, deletion, or fusion of pCCL-CLIC1 and pCCL-CLIC4 plasmids. DNA sequencing was performed to verify the correct sequence before experiments.