*4.6. Evaluation of Protein Quantity by Western Blotting*

A549 cells in 6-well plates or 60 × 15 mm culture dishes were incubated with H2O<sup>2</sup> + LPS with/without BUD, HPβCD, or BUD:HPβCD complex. After incubation, cells were washed with ice-cold PBS and scraped off with a cold scraper in the presence of ice-cold RIPA or Biovision's cell lysis buffer supplemented with protease and phosphatase inhibitor cocktails. Detached cells in lysis buffer were then incubated for 30 min at 4 ◦C with agitation in a 2-mL microcentrifuge tube and centrifuged for 10 min (10,000× *g*, 4 ◦C). The supernatant (whole-cell lysate) was stored at −80 ◦C at least overnight. A quantity of 30 µg of proteins per sample was mixed with 1X NuPAGE LDS sample buffer and 1X NuPAGE sample reducing agent (Thermo ScientificTM, Carlsbad, CA, USA) and heated for 10 min at 70 ◦C. Samples were then electrophoresed on precasted NuPAGE Bis-Tris gels in the presence of MOPS (3-(N-morpholino)propanesulfonic acid) running buffer 1X, transferred to PVDF (Polyvinylidene difluoride) transfer membranes (Thermo ScientificTM, Rockford, IL, USA) in the presence of NuPAGE transfer buffer 1X (Thermo ScientificTM, Carlsbad, CA, USA) and blocked for 1 h in 5% non-fat dry milk in 20 mL of tris-buffered saline 1X containing 0.05% Tween 20 (TBS-T). Membranes were incubated overnight at 4 ◦C with primary antibodies with gentle agitation, washed 3 times with TBS-T, then incubated for 1 h at room temperature with the appropriated HRP-conjugated secondary antibodies. The manufacturer's recommendations were followed for antibody dilutions. After washing 3 times with TBS-T, blots were revealed using the SuperSignal West Pico Chemiluminescent Substrate (Thermo ScientificTM, Rockford, IL, USA), the Fusion Pulse 7 apparatus and Fusion Capt Advance Pulse 7 software. To reveal proteins with similar migration profiles, membranes were washed in TBS-T after the first reveal, and antibodies were stripped with a 10-min bath in RestoreTM Western Blot Stripping Buffer (Thermo ScientificTM, Rockford, IL, USA) and washed again with TBS-T. Then, the Western blot protocol was repeated from the block for 1 h in 5% non-fat dry milk in 20 mL of TBS-T.
