*4.2. CO2 Exposure*

ATII and A549 cells were exposed to 40 (normocapnia, Ctrl) or 120 mmHg (hypercapnia, CO2) CO2 conditions unless otherwise indicated. Normocapnia and hypercapnia media solutions were prepared freshly with DMEM or DMEM/F12, Ham's F12, and MOPS base to obtain a final pH of 7.4 at 40 (5%) or 120 mmHg (15%) of CO2 while maintaining 21% O2 balanced with N2 and were kept overnight in the humidified C-chamber (BioSpherix Ltd., Parish, NY, USA), as described previously [24]. Levels of CO2 in the chamber were controlled by an RO-CO2 carbon dioxide controller (BioSpherix Ltd., NY, USA). Before experiments media pH, pCO2 and pO2 levels were measured using a Rapid-lab blood gas analyzer (Siemens, Erlangen, Germany).

#### *4.3. Western Blot Analysis*

Protein concentrations were determined by using the Bradford assay after lysing the cells. Equal protein concentrations were resolved in 7%–10% polyacrylamide gels and transferred to a nitrocellulose membrane using a semidry apparatus from Bio-Rad (Hercules, Berkeley, CA, USA). Membranes were blocked for one hour in 5% fat-free milk and were subsequently incubated with primary antibodies overnight at 4 ◦C. For some experiments, loading control was performed by staining of the nitrocellulose membranes with Coomassie brilliant blue 250 (Sigma Aldrich, St. Louis, MO, USA). Densitometric analyses were performed by using the Image J software (NIH, Bethesda, MD, USA).

#### *4.4. Cell Surface Biotinylation*

After hypercapnia exposure, PM surface proteins were labeled for 20 min by using 1 mg/mL EZ-Link-NHS-SS-biotin (Pierce Biotechnology, Waltham, MA, USA). Proteins were pulled down with streptavidin-agarose beads (Pierce Biotechnology, Waltham, MA, USA) and analyzed by SDS-PAGE and immunoblotting, as described previously [14].
