*2.9. Immunoblotting for Transcriptional Activity*

To observe the effects of iNPs on transcriptional activity, BMMΦ were seeded at 1.0 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/well in sterile 6-well plates and incubated with 300 µg/mL of the different iNP formulations at 37 ◦C and 5% CO<sup>2</sup> for three hours. Excess iNPs were removed by washing twice with PBS followed by replacing with BMMΦ media. Cells were then challenged with 100 ng/mL LPS for 0.5, 1, or 4 h where indicated, at 37 ◦C and 5% CO<sup>2</sup> before washing twice with PBS and harvested using 300 µL RIPA buffer containing 1% Halt® Protease Inhibitor.

Wells treated with BIRB 796 were made to a concentration of 5 µM and incubated for 15 min prior to LPS induction. BIRB 796 was not washed from the wells. Wells treated with OGM were made to a concentration 10 µM and incubated for 4 h at 37 ◦C and 5% CO2, washed twice with PBS to remove excess OGM, replaced with BMMΦ media, followed by iNP treatment as described above. Wells exposed to UV light were exposed in the cell culture hood for 15 min with the plate lids removed. After exposure, media was exchanged and cells were returned to the incubator and harvested at 0.5, 1, or 4 h after exposure.

Protein lysates were generated using 50/50 sample to 2× SDS-PAGE sample buffer. Proteins were then separated by SDS-PAGE and immunoblotted using the antibodies listed above. ECL was used for detection.
