Protein amount in each sample was detected by RPPA, as previously described (Mueller et al., 2010 (link)). Briefly, samples were washed in PBS twice to remove the fixative, lysed in 40 uL protein extraction buffer (equal volumes of 2-Tris-Glycine SDS Sample Buffer (Invitrogen, Carlsbad, CA), and T-PER Tissue Protein Extraction Reagent (Pierce/Thermo-Fisher, Rockford, IL) plus 1.0% 2-β-mercaptoethanol (Sigma-Aldrich) at an approximate ratio of 1,000 cells/μL. Proteins were denatured by heating for 5 min at 100 C prior to dilution in the microtiter plate. Serial 2-fold dilutions of the lysates were printed in duplicate on glass backed nitrocellulose array slides (Nexterion Slides, Schott, Elmsford, N) in a dilution curve representing undiluted lysate and 1:2, 1:4, and negative control dilutions, using an Aushon 2470 arrayer (AushonBiosystems, Billerica, MA) equipped with 350 μM pins. Each spot was printed with approximately 30.0 nL of lysate/spot. The slides were stored with desiccant (Drierite, W. A. Hammond, Xenia, OH) at −20°C prior to immunostaining.
Printed slides were prepared for staining by treating with 1xReBlot (Millipore, Billerica, MA) for 15 min, followed by 2 × 5 min washes with PBS. Slides were treated for 1 h with blocking solution (1 g of I-block (Applied Biosystems, Bedford, MA), 0.5% Tween-20 in 500 mL of PBS) with constant rocking at room temperature. Each slide was incubated with a single primary antibody at room temperature for 30 min.
Each array was probed with a single polyclonal or monoclonal primary antibody.
Primary antibodies used to investigate 25 endpoints for RPPA are listed in Table2 . Each antibody was subjected to validation by immunoblotting prior to use on the RPPA. Antibody validation criteria included detection of a single band at the appropriate molecular weight in positive control lysates and the absence or significantly reduced presence of a band in negative control lysates by immunoblotting.
The negative control slide was incubated with antibody diluent. Secondary antibody was goat anti-rabbit IgG heavy + chain (1:10,000) (Vector Laboratories, Burlingame, CA). Subsequent protein detection was amplified via horseradish peroxidase-mediated biotinyltyramide with chromogenic detection (diaminobenzidine) according to the manufacturer's instructions (Dako).
Total amount per microarray spot was normalized on total DNA level as previously described (Chiechi et al., 2012 (link), 2013 (link)), and confirmed on total protein and housekeeping protein beta-actin. Total protein staining was performed using Sypro Ruby Protein blot stain (Invitrogen) according to the manufacturer's instructions and scanned with a NovaRay CCD imager (Alpha Innotech, San Leonardo, CA, USA) equipped with a Cy3 filter.
The stability of three normalization analytes (ssDNA, total protein and β-actin) and three protein analytes (Akt Ser473, Akt Thr308, ERK Thr202/Tyr204) was evaluated by geNorm and NormFinder as previously reported (Chiechi et al., 2012 (link)).
Arrays were scanned, spot intensity analyzed with commercial software Image Quant v.5.0, data normalized, and a standardized, single data value was generated for each sample on the array. Additional quality control measures for antibody staining included evaluation of reference lysate staining by visual inspection of scanned images and examination of data analysis results for the positive and negative reference lysates.
Printed slides were prepared for staining by treating with 1xReBlot (Millipore, Billerica, MA) for 15 min, followed by 2 × 5 min washes with PBS. Slides were treated for 1 h with blocking solution (1 g of I-block (Applied Biosystems, Bedford, MA), 0.5% Tween-20 in 500 mL of PBS) with constant rocking at room temperature. Each slide was incubated with a single primary antibody at room temperature for 30 min.
Each array was probed with a single polyclonal or monoclonal primary antibody.
Primary antibodies used to investigate 25 endpoints for RPPA are listed in Table
The negative control slide was incubated with antibody diluent. Secondary antibody was goat anti-rabbit IgG heavy + chain (1:10,000) (Vector Laboratories, Burlingame, CA). Subsequent protein detection was amplified via horseradish peroxidase-mediated biotinyltyramide with chromogenic detection (diaminobenzidine) according to the manufacturer's instructions (Dako).
Total amount per microarray spot was normalized on total DNA level as previously described (Chiechi et al., 2012 (link), 2013 (link)), and confirmed on total protein and housekeeping protein beta-actin. Total protein staining was performed using Sypro Ruby Protein blot stain (Invitrogen) according to the manufacturer's instructions and scanned with a NovaRay CCD imager (Alpha Innotech, San Leonardo, CA, USA) equipped with a Cy3 filter.
The stability of three normalization analytes (ssDNA, total protein and β-actin) and three protein analytes (Akt Ser473, Akt Thr308, ERK Thr202/Tyr204) was evaluated by geNorm and NormFinder as previously reported (Chiechi et al., 2012 (link)).
Arrays were scanned, spot intensity analyzed with commercial software Image Quant v.5.0, data normalized, and a standardized, single data value was generated for each sample on the array. Additional quality control measures for antibody staining included evaluation of reference lysate staining by visual inspection of scanned images and examination of data analysis results for the positive and negative reference lysates.
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