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Recombinant adenoviruses harboring Thbs4, the N-terminal Laminin G (Ad-LamG) domain of Thbs4, the Type III repeat (Ad-T3R) domain of Thbs4, a Thbs4 Ca²⁺-binding mutant containing mutations in six DXDXDG calcium-binding sites within the T3R domain of mouse Thbs4 (Ad-Thbs4-mCa²⁺), constitutively-nuclear ATF6α (Ad-ATF6α-CN, amino acids 1–364), the ER luminal domain of ATF6α (amino acids 448–570) with a C-terminal KDEL ER retention signal (Ad-ATF6α-DN) and β-gal control were previously generated and validated (Brody et al., 2016 (link); Lynch et al., 2012 (link)). Adenoviral LamG, T3R and Thbs4-mCa²⁺ were engineered to contain the N-terminal signal peptide and the coiled-coil domain of Thbs4 to ensure proper intracellular trafficking and assembly and oligomerization in the ER (Brody et al., 2016 (link)). The cDNAs of human Nell2 (Harvard Plasmids, HsCD00331038) and eGFP-tagged VSV-G-ts045 (VSVG-eGFP, Addgene: Plasmid #11912 deposited by Dr. Jennifer Lippincott-Schwartz) were amplified by PCR for insertion into the pAdenoX-CMV vector (Clontech, Mountain View, CA) and transfected into HEK cells to generate recombinant adenovirus following manufacturer’s instructions (Brody et al., 2016 (link); Patterson et al., 2008 (link)).

The Thbs3 cDNA was sub-cloned into the pShuttle-CMV (Agilent Technologies #240007) vector to produce recombinant adenovirus following manufacturer’s instructions. The pShuttle-CMV-Thbs3 construct was used to generate the Thbs3 RGD mutant (5′-cacaggcatctcctcttccattgccatccgtgtcct-3′) using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, #200522). Adenoviruses harboring Thbs4, the endoplasmic reticulum (ER) luminal domain of activating transcription factor 6α (ATF6α, amino acids 448–570) with a C-terminal ER KDEL retention signal (Ad-ATF6α-DN-Myc-KDEL) and β-galactosidase (βgal) expressing control were previously generated and validated^{24 (link)}. The eGFP-tagged VSVG-ts045 (VSVG-eGFP, Addgene: Plasmid #11912) cDNA was amplified by PCR for insertion into the pAdenoX-CMV vector (Clontech, #632269) and transfected into HEK cells to generate recombinant adenovirus following manufacturer’s instructions^{24 (link)}. NRVMs were grown in Medium 199/EBSS (ThermoFisher Scientific, #SH30253FS) supplemented with 2% bovine growth serum (ThermoFisher Scientific, # SH3054103) and 1% penicillin-streptomycin (Cellgro, #30-0002-CI). NRVMs were then infected with adenovirus for 2 h and then fresh media was applied. Cells were harvested 24 to 72 h post-infection.