Quantification of Protein Expression via Western Blot

Protein extraction was performed as previously described,^{37 (link)} and protein contents were quantified by BCA assay according to the manufacturer’s instructions (Thermo Fisher 23221). For western blotting, 20 µg were loaded per well on 7.5% precast TGX gel (Bio-Rad 4561026). Proteins were then transferred on PVDF membrane (Bio-Rad 10026933). The primary antibodies used for western blotting were as follows: CA IX (Novus Biological, 1:1000), NHE1 (Novus Biological, 1:1000) and vinculin (Santa Cruz Biotechnology 73614, 1:5000). Secondary antibodies were purchased from Sigma (anti-mouse IgG: A4416; anti-rabbit IgG: A6154). Clarity-ECL substrate was used to reveal bands (Bio-Rad 1705060) and protein expression was finally determined by quantification of band intensity using ImageLab (Bio-Rad). Real-time PCR method is described in Supplementary Material.