N-terminal, HA-epitope tagged, human CCM2 CCCTdup was cloned into the pLVX-TRE3G lentiviral vector (Takara Clontech) and lentivirus was generated from co-transfection with packaging plasmids in HEK293T cells as previously described12 (link). HUVECs were co-infected with CCM2 CCCTdup along with Tet-On 3G lentiviruses and varying doxycycline amounts were added as previously described12 (link). Cells were harvested 48 hours post-doxycycline addition. Collected cells were pelleted, resuspended, then divided in half. One half was used for total RNA isolated by TRIzol (Life Technologies), and the other half was used for protein extracted by gentle lysis buffer as described above. cDNA was generated from 500 ng of total RNA using the Superscript III Reverse Transcriptase (Invitrogen). mRNA levels of the CCM2 CCCTdup lentivirus were assessed by qPCR using both N-terminal and C-terminal human CCM2 qPCR primers as follows:
Western blotting was performed as previously described with 75 μg protein from HUVEC lysate for each condition.
Western blotting was performed as previously described with 75 μg protein from HUVEC lysate for each condition.