Table 1.
Description of antibodies and western blot conditions
| Prep | Protein loading | Denaturation | Blocking (90 min, RT) | PAK (overnight 4°C) | SAK (90 min, RT) | |
|---|---|---|---|---|---|---|
| SGLT1 | BBM | 12 μg | 40°C, 15 min | 5% MP/TBST | 1:2000 | 1:20,000 |
| pSGLT1 | BBM | 12 μg | 40°C, 15 min | 5% MP/TBST | 1:2000 | 1:15,000 |
| GLUT2 | CM | 20 μg | 70°C, 10 min | 2,5% MP/TBST | 1:2000 | 1:30,000 |
| β2‐receptor | CM | 12 μg | 70°C,10 min | 5% MP/PBST | 1:200 | 1:10,000 |
| PKA substrate | Cyto | 9 μg | 95°C, 5 min | 5% MP/TBST | 1:1000* | 1:2000 |
| AMPK | Cyto | 9 μg | 95°C, 5 min | 5% MP/TBST | 1:1000* | 1:2000 |
| pAMPK | Cyto | 9 μg | 95°C, 5 min | 5% MP/TBST | 1:1000* | 1:2000 |
Antibody‐specific parameters are indicated for used preparations (Prep) (BBM, brush border membrane; CM, crude membrane; cyto, cytosol) protein loading, denaturation conditions, blocking conditions (MP/TBST, MP/PBST = dry milk in TRIS or phosphate‐buffered saline with 0.1% Tween 20) and incubation times as well as dilutions of primary (PAK) or secondary antibodies (SAK). Antibodies were diluted in the respective blocking solution with exception of PAKs indicated by “*” which were diluted in 5% bovine serum albumin/TBST. pSGLT1 was detected using SuperSignal® West Femto Maximum Sensitivity Substrate instead of SuperSignal® West Dura Extended Duration Substrate (both Thermo Scientific).