Bioplex 200 multiplexing analyzer system

Cell lysates to be analyzed for protein expression by western blotting were harvested in protein lysis buffer and prepared as previously described [51 (link)]. Antibodies used were specific for human proteins RIG-I (Adipogen, AG-20B-0009-C100), ISG56 (from G. Sen at Cleveland Clinic Foundation, Cleveland), Mx-1 (W. Lai, Antibody Production Core, UT Southwestern Medical Center), MAVS (Enzo Life Sciences, ALX-210-929-C100), MDA5 (IBL America, 29020), HTNV nucleocapsid 76–118 (BEI resources, NR-12152), and actin (Santa Cruz, SC-1616). All secondary antibodies were obtained through Jackson ImmunoResearch and visualized on a Licor Odyssey CLx imager. Images were cropped and uniformly modified using the Image Studio imaging software and Adobe Illustrator. Densitometry was performed using ImageJ software (NIH) and calculated as the relative density of HTNV nucleocapsid protein over actin. Cytokine/chemokine analysis was performed on UV-treated, homogenized mouse tissues using the Milliplex MAP mouse cytokine/chemokine magnetic bead panel kit (Millipore, MCYTOMAG-70k) run on a Bio-Rad Bioplex 200 Multiplexing Analyzer System according to manufacturer instructions. Results were analyzed and graphed using Prism 5 software (GraphPad).

Antibody measurements were acquired using a Bio-Plex^® 200 Multiplexing Analyzer System from Bio-Rad for all non-magnetic coupled beads (Bio-Plex^® 200System, Bio-Plex^® high-throughput fluidics system, microplate platform and a computer with the Bio-Plex^® manager software v.5.0). Washing steps were carried out on a Bio-Rad Aurum vacuum manifold.
For all magnetic coupled beads a MAGPIX^® Multiplexing System from Millipore was used (MAGPIX^® System and the Xponent software V.4.2). Washing steps were carried out using a magnetic plate washer from BioTek Instruments (BioTek ELx50). A Bio-Rad Sure Beads magnetic rack was used during the coupling process.
Plates were incubated on a Ratek Platform shaker (Microtiter/PCR Plate Shaker). A Vortex Sonicator (Branson 2200), a BioSan Vortex V-1 plus and a Table centrifuge (Eppendorf Centrifuge 5424) were also used during the coupling process.

Bioluminescence quantification of p50/65 heterodimer binding activity was performed in AEC2s using our published lentiviral NFκB signaling reporter vector (51 (link)) with methods for transduction of iAEC2s, sorting, and bioluminescence measurements detailed in our prior publication (36 (link)). Day 258 W308R and cW308 patient iAEC2s, and day 158 E690K and cE690 iAEC2s were transduced for biolumescence profiling. To quantify cytokine secretion, supernatants were harvested from iAEC2s after 2D culture and analyzed through a human magnetic Luminex assay (R&D Systems) on Bio-Plex 200 multiplexing analyzer system (Bio-Rad), with cytokine list and methods detailed in Supplemental Materials.