Quantification of carboxy-terminal fragment levels

Quantification of carboxy-terminal fragment levels

1. Seed 5x10⁵ LLC-PK1 cells on 6-well plates 
2. 24h after, change medium to medium without serum (alpha-MEM supplemented containing 100 U/ml P/S and 2 mM glutamine)
3. After 10 hours of starting depletion, replace the medium by a serum-free medium containing 10 μM of the gamma-secretase inhibitor DAPT (or vehicle).
4. 14 hours after starting treatment with DAPT, change medium by serum-free medium containing 2.5 or 20 ng / ml of TGFb with or without DAPT plus MG132 10 uM by 10h.
   To complete 24h of treatment with or without DAPT and 10 hours of treatment with TGFb plus MG132.
5. Subsequently lyse cells in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 150 mM EDTA pH 8.0, 1% NP40, 0.5% Deoxycholate, 0.1% SDS) plus 2mM PMSF, 1 mM pepstatin, 2 μM antipain, 1 μM leupeptin, 1mM glycerophosphate, 1 mM orthovanadate and 5 mM NaF for 40min at 4˚C
6. Remove cells using a cell scraper, and eliminate the nuclei by centrifugation at 20,000x g for 5 min at 4˚C. 
7. After, measure protein concentrations with a BCA protein assay kit and run 60 ug of each lysate on a 15% bis-acrylamide gel in reducing conditions.
8. Lately, transfer the proteins to a PVDF membrane of 0.2 uM by wet transfer system (350 mA, 3h).
9. Incubate blot, successively with the corresponding primary antibodies (mouse anti- tubulin and rabbit polyclonal anti-megalin tail) and the corresponding horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG. Detect the immunoreactive proteins with the ECL system.
   • Carboxy-terminal fragments (CTF) are detected using the polyclonal antiserum to the recombinant human megalin cytoplasmic domain (anti-megT) previously described in Marzolo et al 2003.
   • Tubulin is detect using anti-ß-tubulin antibody (Millipore)
10. Perform densitometric analysis using ImageJ 1.45s software