*4.5. Co-Immunoprecipitation*

To assess protein-protein interactions, co-immunoprecipitation was used. AEC were exposed to normocapnia or hypercapnia, washed in PBS, and lysed in immunoprecipitation lysis buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA, 2 mM EGTA, 5% glycerol, protease and phosphatase inhibitors). Afterward, cells were scraped and centrifuged and cell lysates containing 150–300 μg of proteins were incubated with specific antibodies for Na,K-ATPase subunits, GFP, 2,4-DNPH, or IgG negative control and A/G agarose beads (Santa Cruz, Heidelberg, Germany) overnight at 4 ◦C. Precipitated complexes were eluted from the beads by adding 2X Laemmli buffer, heated at 60 ◦C for 20 min, and then subjected to SDS-PAGE and Western immunoblotting.

### *4.6. Antibodies and Chemical Compounds*

The following antibodies and reagents were used: mouse anti-Na,K-ATPase β-subunit (clone M17-P5-F11) (Thermo Scientific, Rockford, IL, USA), rabbit anti-GFP (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Na,K-ATPase α-subunit (Merck Millipore, Darmstadt, Germany), rabbit anti-β-actin (Sigma Aldrich, St. Louis, MO, USA), mouse anti-transferrin receptor (Invitrogen, Rockford, IL, USA), rabbit anti-PDI (Cell Signaling, Danvers, MA, USA), rabbit anti-GM130 (Cell Signaling, Danvers, MA, USA), rabbit anti-calnexin (Abcam, Cambridge, UK), rabbit anti-BiP (Cell Signaling, Danvers, MA, USA), HRP-conjugated anti-mouse and anti-rabbit IgG (Cell Signaling, Danvers, MA, USA), and Alexa Fluor 488 and 594 conjugated anti-rabbit, anti-mouse (Thermo Scientific, Eugene, OR, USA). α-ketoglutaric acid was obtained from Sigma Aldrich, St. Louis, MO, USA.

#### *4.7. Immunofluorescent Microscopy*

After hypercapnia exposure cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked by 3% bovine serum albumin (BSA), and incubated overnight with primary antibodies at 4 ◦C. Immunofluorescent images were captured by using a Carl Zeiss Axio Observer Z1 microscope (Carl Zeiss, Wetzlar, Germany).

#### *4.8. Isolation of Total, Cytosolic Endoplasmic Reticulum and Golgi Cellular Fractions*

Isolation of ER and Golgi was performed by adapting a previously described protocol [54]. After normocapnic and hypercapnic exposure cells were collected, homogenized, and centrifuged 10 min at 1400× *g* following supernatant centrifugation for 10 min at 15,000× *g*, 4 ◦C. Next, a small amount of the supernatant was aspirated and labeled as "total protein" and the rest was loaded on a sucrose gradient (2.0 M, 1.5 M, and 1.3 M) and centrifuged for 70 min at 152,000× *g*. Afterward, the upper 1 mL of the solution was withdrawn and labeled as "Cytosol." The ER fraction was collected from the large band at the interface of the 1.3 M sucrose gradient layer, resuspended in lysis buffer, and centrifuged for 45 min at 126,000× *g*, 4 ◦C. Afterward, the pellet was collected, resuspended in PBS (pH 7.4), and further analyzed by a Western blot. Since ER and Golgi are morphologically linked and have almost the same density during ultracentrifugation, the obtained ER proteins additionally contained Golgi complexes and, in some experiments, this fraction was named "ER, Golgi".
