Cell lysates were boiled in Laemmli sample buffer and were separated by SDS-PAGE, transferred to nitrocellulose filters, probed with the antibody of interest, and developed using ECL (Amersham Biosciences, Pittsburg, PA, USA).
Then, samples were reduced with β-mercaptoethanol in Laemmli sample buffer and were separated by SDS-PAGE electrophoresis.
Proteins bound to the beads were eluted with 2 × SDS-PAGE sample buffer and were separated on an SDS-PAGE gel.
The samples of surnatants were thereafter diluted with reducing sample buffer and were separated by electrophoresis on a 10% SDS-PAGE gel (20 μg protein per lane loaded).
The retained peptides were washed with the same buffer and were separated on a PicoFrit Analytical Columns (ProteoPep II C18, 300 Å, 5 μm, 50 μm × 100 mm, tip ID=10 μm, New Objective, Inc).
Eluates were vacuum dried and resuspended in protein sample buffer (1×MES buffer+1×protein loading buffer, Life Technologies) and were separated by SDS-PAGE on a 10 20% NuPAGE gradient gel (Life Technologies).
Briefly, proteins were diluted in 50 mM Bis-Tris buffer pH 7.0 and were separated on 3-12% Bis-Tris gels (Invitrogen, Paisley, UK) in the presence of a final concentration of 0.05% Triton x-100.
Equal volume of protein (20 μL) samples were incubated for 3 minutes at 95°C in a loading buffer (5 μL), and were separated on a 10% SDS polyacrylamide gel.
Briefly, human ADSC samples and epithelial-like cells were selected using the same method, lysed in RIPA buffer and protein lysates and were separated on 8 % SDS polyacryamide gel by electrophoresis.
Inclusion bodies containing Trim17 were dissolved in Laemmli buffer and Trim17 was separated by preparative SDS PAGE.
