EIF4EBP1 protein, human

The same number of cells (25,000-35,000) from each population to be analyzed were sorted into Trichloroacetic acid (TCA) and adjusted to a final concentration of 10% TCA. Extracts were incubated on ice for 15 minutes and spun down for 10 minutes at 16.1 rcf at 4°C. The supernatant was removed and the pellets were washed with acetone twice then dried. The protein pellets were solubilized with Solubilization buffer (9 M Urea, 2% Triton X-100, 1% DTT) before adding LDS loading buffer (Invitrogen, Carlsbad, CA). Proteins were separated on a Bis-Tris polyacrylamide gel (Invitrogen) and transferred to a PVDF membrane (Millipore, Billerica, MA). Antibodies were anti-Lkb1 (#3047), anti-phospho-AMPKα (Thr172) (#2535), anti-AMPKα (#2532), anti-phospho-Acetyl-CoA Carboxylase (Ser79) (#3661), anti-phospho-S6 (#2215), anti-phospho-4EBP1 (#2855), anti-phospho-eIF4G (#2441) (all from Cell Signaling Technology, Danvers, MA) and anti-ß-actin (A1978, Sigma).

Using a 2470 Aushon arrayer outfitted with 185-μm pins, experimental samples were printed onto nitrocellulose covered slides (GRACE Bio-Labs; Sartorius Stedim Biotech) along with standard curves and internal controls. Samples, reference standards and internal controls were printed in three or four technical replicates on each slide. To verify that each sample was in the linear dynamic range of the assay, a BSA serial dilution curve was added to the array to estimate the protein concentration of each sample.
Immunostaining was performed on an automated DAKO system using a commercially available Catalyzed Signal Amplification (CSA) Kit (Dako). Samples included in the training set were probed using primary antibodies targeting AKT (S473) (Cell Signaling Technology, catalog no. 9871; dilution 1:100), and the mTOR downstream substrate p70S6K (T389) (Cell Signaling Technology, catalog no. 9205; dilution 1:100). The validation set was probed with AKT (S473) (Cell Signaling Technology, catalog no. 9871; dilution 1:100), mTOR (S2448) (Cell Signaling Technology, catalog no. 2971; dilution 1:100), and two downstream substrates: 4EBP1 (S65) (Cell Signaling Technology, catalog no. 9451; dilution 1:100), and S6 Ribosomal Protein (S6RP) (S235/236; Cell Signaling Technology, catalog no. 4858; dilution 1:100). Each antibody was rigorously validated on a panel of cell lines and human samples using conventional Western blotting technique and tested on the arrays to assure the linear dynamic range of the analytes was captured (35 (link)).
Signal detection was achieved using a biotinylated goat antirabbit secondary antibody (1:7,500 and 1:5,000 for the discovery and validation set, respectively; Vector Laboratories) coupled with a biotinyl-tyramide–based amplification system (34 ). The IRDye 680RD Streptavidin (LI-COR Biosciences; dilution 1:50 in PBS supplemented with 1% BSA) or the Cy5 Strepatavidin (KPL; dilution 1:100 in PBS supplemented with 1% BSA) fluorescent detection systems were used for the discovery and validation set, respectively. Finally, to quantify the amount of protein in each sample, selected slides were stained with Sypro Ruby Protein Blot Stain (Molecular Probes) following manufacturer’s recommendation (34 ).
A laser scanner was used to acquire antibody and Sypro Ruby stained slides (TECAN or Genepix 4200 AL, Molecular Devices). Images were analyzed using the commercially available microarray software MicroVigene Version 5.1.0.0 (Vigenetech) or Genepix Pro 6.1 (Molecular Devices). Final intensity values were generated after: (i) subtraction of background and unspecific binding generated by the secondary antibody; (ii) normalization to the corresponding amount of protein derived from the Sypro Ruby–stained slides; and (iii) average of the technical replicates. Experimental sample values were interpolated from the reference standards using standard linear interpolation techniques. Each patient value was compared to the reference population and scored on a categorical scale (above or below the cut-off point).