Washed complexes/lysates, dissolved in Laemmli buffer, were resolved in 10%-12 10%-12AGE.
Briefly, extracts prepared in SDS loading buffer were resolved in SDS/10% PAGE gels and transferred to PVDF membranes.
Briefly, urine resuspended in SDS loading buffer was resolved on a 7.5% polyacrylamide gel in Tris glycine buffer and in gel protein staining was carried out with Coomassie Blue (Euromedex, Souffelweyersheim, France).
Reduced protein in Laemmli sample buffer was resolved using SDS-PAGE and transferred to Immobilon-P 0.45 μm PVDF membrane (EMD Millipore, Billerica, MA).
For WB of IGF-I (Fig. 5), 1.0 μL plasma samples diluted in reducing Laemmli buffer was resolved by PAGE (4 20% Criterion TGX Bio-Radd) and transferred to PVDF membranes.
20 μg to 30 μg of total protein in SDS sample buffer was resolved by SDS-PAGE (10% or 12.5%) and transferred to PVDF membrane using a semi-dry transfer system (Hoepher SemiPhor).
The protein flow through was resolved in RIPA buffer with 10% SDS.
The pellet produced by TCA precipitation was resolved in RIPA buffer containing a protease inhibitor cocktail (Sigma-Aldrich) and PMSF (500 1 1).
Brain, liver, and spleen homogenates (10%) prepared in lysis buffer were resolved by SDS-PGE and subjected to western blotting using specific antibodies as described in previous reports [30].
Cell lysates extracted in RIPA buffer were resolved by 3 8% SDS-PAGE.
