Cell culture conditions and cell lysis
Human colon cancer cell lines HCT15 (CCL-225), HCT116 (CCL-247), H508 (CCL-253), SW620 (CCL-227), HT29 (HTB-38), RKO (CLR-2577), and SW480 (CCL-228) as well as the breast cancer cell lines HCC1954 (CRL-2338), MDA-MB-231 (HTB-26), BT-474 (HTB-20), SK-BR-3 (HTB-30), MCF-7 (HTB-22), and T47D (HTB-133) were obtained from American Type Culture Collection (Manassas, USA). Breast cancer cell lines were cultivated under conditions recommended by ATCC (ATCC serum and medium annotation, http://www.lgcpromochem-atcc.com). All colon cancer cell lines were cultivated in RPMI 1640 medium (ATCC) containing 10% FCS and 1% Pen/Strep (both Invitrogen, Karlsruhe, Germany). Cell lines were grown until 90% confluence and split 3 times per week. For lysis, cultured cells were incubated with trypsin (Sigma, Munich, Germany) and lysed in 1 ml M-PER (Pierce, Rockford, USA) containing 2 mM ortho-sodium-vanadate (Sigma), 10 mM NaF, and Complete mini protease inhibitor. The protein concentration was determined in duplicate by BCA Protein Assay (Pierce). Protein lysates were stored at -80°C until use.
Prostate tissue samples were collected at Martini-Clinic, Prostate Cancer Center (University Medical Center Hamburg-Eppendorf, Germany). Samples were acquired during radical prostatectomy and sectioned with a microtome. One slice of prostate tumor tissue was processed per experiment. Mouse liver tissue and collected prostate tissue were lysed with modified T-PER buffer (Pierce). 10 μl of lysis buffer were used per mg of tissue sample. Samples were homogenized 4 minutes with a tissuelyser (Qiagen, Hilden, Germany). Cell debris was subsequently pelleted (12 minutes, 13000 rpm) and the supernatant was passed through a Qiashredder tube (Qiagen).
Printing of protein microarrays
Protein lysates and spike-in experiments with recombinant His-tagged JNK (Invitrogen) were serially diluted with protein lysis buffer. To prepare samples for inkjet-spotting using the Sprint (ArrayJet, Roslin, Scotland) an equal volume of spotting-buffer (50% (v/v) Glycerol and 0.05% (w/v) Triton × 100 in ddH20) was added to each sample resulting in a final glycerol concentration of 25%.
A pin tool spotter, the 2470 Arrayer (Aushon, Billerica, MA, USA), was employed to print serial dilutions of cell line lysates (SW480, HCT15, HCT116, SW620, HT29, HCC1954, MDA-MB-231, BT-474, SK-BR-3, MCF-7 and T47D). Dilution series comprised 14 steps and were produced by diluting with lysis buffer by 33% per step. Samples were printed directly after adding Tween20 to a final concentration of 0.05% (v/v).
Samples were always printed onto nitrocellulose coated glass-slides (Grace Biolabs, Bent, OR, USA). Each spot corresponded to a final drop volume of 0.3 nl (pin tool spotter) or 0.6 nl (non-contact inkjet printer). The spot-to-spot distance was set to 320 μm and the spot diameter was 140 μm. Slides were stored at 4°C and used within a week.
Near infrared target protein detection on microarrays
Slides were blocked with a mixture of 33% Odyssey blocking buffer (LI-COR, Lincoln, USA), 1% BSA, and 0,02% NP40 in PBS overnight at 4°C. Primary antibodies were diluted in a buffer with background-reducing components (Dako, Glostrup, Denmark). Slides were incubated for 2 h with primary antibodies and subsequently immersed in wash buffer (1x PBS, 0.02% NP40 and 0.02% SDS) four times for 5 minutes. Next, slides were incubated with Alexa680-conjugated secondary antibody (Invitrogen, dilution 1:8,000) for 30 minutes. Washing was performed as described above. All washing and incubation steps were carried out at RT with gentle shaking. Finally, slides were rinsed in water and air-dried at room temperature.
The following detection antibodies were employed for western blotting and RPPA-based analysis: JNK (BD Biosciences, San Jose, USA), CDK4, Cyclin D1, GSK, PP2AA, PP2AB, PDK1, beta-Catenin, RB, Smad 2/3, SRC (Cell Signaling Technologies, Danvers, USA), ERK 1/2, NF-κB, PCNA, Stat3 (Santa Cruz, Santa Cruz, USA), KLK3 (Sigma), JNK, PKC, PLCγ (Abcam, Cambridge, USA), and pERK (R&D, Minneapolis, USA).
Antibody-mediated signal amplification on microarrays
Incubation with primary antibodies was carried out as described in the previous section. All working steps for antibody-mediated signal amplification were integrated into an automated procedure. A robotics protocol was established to increase and simplify the throughput. The 96-channel head robot Biomek FXP (Beckmann Coulter, Harbor Boulevard, USA) was used for all assay steps thus reducing hands-on time and minimizing experimental variation.
Anti-rabbit Alexa680-labeled (raised in goat) and anti-goat Alexa680-labeled (raised in rabbit) antibodies (both Invitrogen) were applied consecutively in a total of four cycles (dilution 1:8,000). Anti-mouse Alexa680 labeled (raised in goat) antibody was used in the first cycle for the detection of primary antibodies raised in mouse. Secondary antibodies employed for signal amplification were derived from commercial sources and of highest purity. Slides were washed four times for 5 minutes between automated incubation steps. Each secondary antibody was incubated for 30 minutes.
Western Blot detection
SDS-Page and Western immunoblotting analyses were performed using 5 to 20 μg protein lysate. Standard near infrared detection was applied as described elsewhere . The protocol for antibody-mediated signal amplification was adapted to a larger volume. Tyramide signal amplification was carried out in parallel to compare directly between both amplification methods. TSA Kit #11 (Invitrogen) was employed according to the manufacturer's instructions. Endogenous peroxidase was treated with 3% H2O2 for one hour. A 1:250 dilution of the HRP conjugated secondary antibody was applied. Peroxidase catalyzed signal amplification was carried out for 10 minutes and Streptavidin-Alexa680 (Invitrogen) was incubated for 20 minutes.
Slides were scanned with the Odyssey NIR scanner (LI-COR). Image analysis was carried out with GenePix-Pro 5.1 (Axon Instruments, Sunnyvale, USA). Spot intensity was corrected for background and noise due to unspecific antibody binding. Mean and SD were calculated for all replicates. Data sets were analyzed within the statistical computing environment R.
The detection limit was determined as follows: First, the signal intensity mean of a certain dilution step has to be greater than the sum of 2 x standard deviation (SD) of S0 and the mean of S0 with S0 corresponding to the lowest concentration of a dilution series. Thus, S0 was considered as background signal  ensuring a significant difference of signals from background. Second, in a dilution series signals must increase continuously with increasing proteins concentrations. The lowest concentration of a certain serial dilution complying with both requirements is defined as limit of detection (LOD). To sum up, the detection limit corresponded to the smallest concentration of a certain serial dilution that can be distinguished from the experimental background as well as from the next higher concentration. A detection limit is a specific measure for a specific detection antibody, detection method, and a specific sample. The curve fitting model and the normalization approach are described in detail in Additional file 1.