Monoclonal antibody against gapdh

Whole cell extracts from cultured cells were prepared using radioimmunoprecipitation assay buffer (Thermo Scientific, Rockford, IL) containing protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein quantification was done using a BCA protein assay kit (Thermo Scientific) according to the manufacturer's instructions. Total protein (20 μg) was loaded onto 4–12% bis–tris gels with 3-(N-morpholino) propanesulfonic acid buffer and separated by a NuPAGE electrophoresis system (Invitrogen). Protein was transferred to Invitrogen™ polyvinylidene difluoride and immunoblotting was carried out according to standard protocols. Monoclonal antibody against PMS2 was purchased from Origene Technologies (Rockville, MD) and monoclonal antibody against GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) was used to confirm equal loading. The membrane was washed and then incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technology). Protein complexes were visualized with the Echo-chemiluminescence (ECL) Detection System (GE Healthcare, Little Chalfont, UK) using the Chemidoc Imaging System (Bio-Rad Laboratories, Hercules, CA).

HEK293 cells transfected with cDNA encoding SLC4A11 v2, v3 and empty vector were lysed as explained above. v2 lysate (10 µg protein), v3 lysates (5 µg, 10 µg and 20 µg protein), vector control lysate (20 µg protein) and pooled human corneal endothelial cell lysate (20 µg protein) were processed on immunoblots in duplicates. Blots were probed with SLC4A11-common antibody and SLC4A11-v3 antibody, respectively followed by Donkey anti-rabbit secondary antibody and developed. GAPDH, used as a loading control, was detected with monoclonal antibody against GAPDH from Santa Cruz Biotechnology and secondary antibody was sheep anti-mouse (GE Healthcare Bio-Sciences Corp. (Piscattaway, NJ, USA). Band intensities (B.I.) were determined by densitometry. The ratio of v3 detection by anti-common and anti-v3 antibodies was determined (see Supplementary Fig. S1). This detection ratio (anti-common/anti-v3) was used to calculate the abundance of v2 and v3 in endothelial lysates as follows: Total SLC4A11 expression (detected with anti-common antibody) = v2 + v3. To convert the abundance of v3 detected by anti-v3 to the amount detected with anti-common, v3 = B.I._α-v3 × Detection ratio. So, v2 = Total SLC4A11 expression (detected with anti-common antibody) - v3 and thus v2 = Total SLC4A11 expression (detected with anti-common antibody) - (B.I._α-v3 × Detection ratio).

Proteins of liver SREBP-1c, PPARα and FAS were determined by western blotting according to the commercially available kits (Key GEN, China) under the standard procedure. The protein contents of the extracts were determined by bicinchoninic acid (BCA) assay (Beyotime, China). 20 µg protein was loaded and separated through SDS-PAGE and transferred onto PVDF (Millipore, Boston, MA). After keeping with 5% skim milk for 1 h, the rabbit polyclonal antibodies against SREBP-1c, PPARα, FAS, Histone 2H.X (1:1,000; Santa Cruz, Santa Cruz, CA) and monoclonal antibody against GAPDH (1:2,000; Santa Cruz) were added and the membranes were subsequently incubated overnight at 4°C; Subsequently, incubated with HRP-conjugated secondary antibody (1:5,000) for 1 h. The relative SREBP-1c, FAS and PPARα protein-levels were analyzed using the Quantity one.