Protein | Prep | Denaturation | µg/lane | Primary antibody | Secondary antibody |
---|
SGLT1a | AM | 40 °C, 15 min | 10 µg | 1:2000 | 1:10,000h |
pSGLT1b | AM | 40 °C, 15 min | 10 µg | 1:200 | 1:10,000i |
ASCT1c | AM | 95 °C, 7 min | 10 µg | 1:1,000 l | 1:2000j |
PepT1d | AM | 95 °C, 7 min | 10 µg | 1:500 l | 1:2000k |
GLUT2e | CM | 70 °C, 20 min | 10 µg | 1:2000 | 1:20,000h,m |
Na+/K+-ATPasef | CM | 70 °C, 20 min | 10 µg | 1:10,000 | 1:20,000k |
Hif-1αg | CY | 95 °C, 5 min | 10 µg | 1:200n | 1:2000i |
- AM apical membrane, CM crude membrane, CY cytosol
- aRabbit-anti-SGLT1 (ab14686, Abcam, Cambridge, UK)
- bCustom-made (Perbio Science, Bonn, Germany; epitope: KIRKRApSEKELMI)
- cRabbit-anti-ASCT1 (#ANT-081, alomone labs, Jerusalem, Israel)
- dMouse-anti-PEPT1 (373,742, Santa Cruz, Dallas, TX, USA)
- eRabbit-anti-GLUT2 (ABIN310208, Antibodies-online, Aachen, Germany)
- fMouse-anti-Na+/K+-ATPase (ALX-804–082, Enzo Life Sciences, Farmingdale, NY, USA)
- gRabbit-anti-Hif-1α (#10,006,421, Cayman chemical, Ann Arbor, MI, USA)
- hGoat-anti-rabbit HRP (A9169, Sigma-Aldrich, St. Louis, MO, USA)
- iGoat-anti-rabbit HRP (#7074, Cell Signaling Technology, Danvers, MA, USA)
- jm-IgGκ BP (516,102, Santa Cruz, Dallas, TX, USA)
- kGoat-anti-mouse (A2304, Sigma-Aldrich, St. Louis, MO, USA)
- lDiluted in BSA (5%) instead of 5% milk/TBST
- mDiluted in 2.5% milk/TBST
- nDiluted in 2.5% milk/PBS