Speedy lentivirus purification kit

Human hCPEC (ScienCell Research Laboratories) were grown in Epithelial Cell Medium purchased from the same provider. Mpdz was knocked down by transduction with lentivirus expressing a validated shRNA (clone TRCN0000349430, MilliporeSigma) or a non‐targeting shRNA (pLKO.1‐puro shRNA Control, MilliporeSigma). The virus was produced in HEK293 cells (American Type Cell Culture) that were transfected (Fugene HD, Promega) with packaging and coat protein plasmids (pCMVR8.74 and pMD2.G, respectively, Addgene). Lentivirus particles were concentrated from the cell culture medium by centrifugation (Speedy Lentivirus Purification kit, ABM). hCPEC were lysed 3 days after lentivirus transduction in RIPA buffer (Thermo Fisher Scientific (TFS)) supplemented with protease inhibitor cocktail tablets (Roche), and immunoblotted with LDLR antibody (MilliporeSigma) diluted 1:1,000. LDLR band density was quantified by an ImageJ plugin and normalized relative to the load‐control band density (antibody to β‐actin, MilliporeSigma diluted 1:2,000).

293T/17 cells were cotransfected with shRNA expressing pLKO.1 lentiviral construct or protein expressing pSIN lentiviral construct plus 2 helper plasmids, pMD.G and pCMVΔR8.91, using Lipofectamine 2000. For shRNA expressing lentiviruses, culture supernatants containing lentiviral particles were harvested and stored at 80°C. For protein expressing lentiviruses, culture supernatants were further concentrated by Speedy Lentivirus Purification kit (LV999, abm). After lentiviral transduction, cells were selected by 10 μg/mL puromycin (InvivoGen) for stable cell line establishment.