Goat anti human aat

Western blotting was carried out following the previous protocol [29 (link)]. After blocking, the membrane was incubated with the primary antibody [goat anti-human gp120 (Abcam), goat anti-human gp41 (Abcam), goat anti-human AAT (Bethyl), mouse anti-human CD4 (Immunotech), mouse anti-human CCR5 (Pharmingem), mouse anti-human CXCR4 (R&D), or rabbit anti-human β-actin (Abcam)]. Next, secondary antibody [anti-goat IgG-HRP (Chemicon international), anti-mouse IgG-HRP (Chemicon international), or anti-rabbit IgG-HRP (Chemicon international)] was used to link to the primary antibody and then developed using ECL Advance Western Blotting Detection Kit (GE Healthcare).

For all ELISAs (human AAT, human PiZ, and c-myc), high binding 96-well plates (Immulon4; Dynatech Laboratories) were coated with the relevant capture antibody in 100 μL of Voller's buffer overnight at 4°C. All subsequent incubations were done at room temperature for 1 h and plates were washed with phosphate buffered saline containing 0.05% Tween 20. Detection antibody was used at 1:5000 goat anti-hAAT-horseradish peroxidase (HRP, Cat#A80-122P; Bethyl Laboratories, Inc.).
Human AAT ELISA: As previously described,^{31 (link)} blocking was done with 1% milk; 1:000 capture antibody goat anti-human AAT (Cat#A80-122A; Bethyl Laboratories, Inc.) was used. Human plasma-purified AAT was used to create the standard curve (Cat#16-16-011609; Athens Research and Technology).
Human PiZ-AAT ELISA: Blocking was done with 3% bovine serum albumin (BSA); 1:000 dilution custom capture antibody was produced in our laboratory (UMMS-PiZ-mAb). In-house standard was used to create the standard curve.
c-myc ELISA: Blocking was done with 3% BSA; 1:1000 c-myc capture antibody (Cat#GTX30518; GeneTex, Inc.) was used. In-house standard was used to create the standard curve.