Lowry kit

Platelets of IRAG2-WT and IRAG2-KO or lacZ × IRAG2-KO mice were isolated as described before [9 (link)]. Briefly, mice were euthanized with CO₂ and blood was drawn by cardiac puncture into 200 µL Alsever’s solution (Sigma Aldrich^®, Taufkirchen, Germany), followed by mixing with 500 µL of buffer B (20 mM HEPES, 138 mM NaCl, 2.9 mM KCl, 1 mM MgCl₂, 0.36 mM NaH₂PO₄, pH 6.2). After centrifugation of the mixture at 70× g for 15 min (min) at room temperature (RT), supernatant was collected and centrifuged at 600× g for 5 min at RT. The resulting platelet pellet was resuspended in buffer B (pH 7.4) for platelet aggregation and ex vivo phosphorylation experiments. For Western Blot experiments, (co-)immunoprecipitation and in vitro phosphorylation, platelet pellet was homogenized directly after isolation with 2% Lubrol-buffer (2% nonaethylene glycol monododecyl ether, 150 mM NaCl, 20 mM Tris in ddH₂O, pH 8.0), containing protease inhibitors (1 mM benzamidine, 0.5 µg/mL leupeptin, 300 µM PMSF) and 1× PhosSTOP (Roche, Mannheim, Germany). The homogenate was centrifuged at 18,000× g for 15 min at 4 °C to remove cell debris and supernatant was collected and stored at −80 °C until further experiments were conducted. Protein concentration was detected using a Lowry kit (Bio-Rad Laboratories, Inc., Munich, Germany).

The anti-TM4SF4 antibody for the Western blot analysis was purchased from Sigma-Aldrich. Antibodies against Sox2, Phospho-AKT (Ser473), AKT, Phospho-NF-κB p65 (Ser536), NF-κB, Phospho-IGF1Rβ (Tyr1131), IGF1Rβ, integrin αV, CD44, and β-actin (Cell Signaling Technology, Danvers, MA, USA); Twist, c-Myc, Cyclin D1, β-catenin (Santa Cruz, Dallas, TX, USA), E-cadherin, and N-cadherin (BD Biosciences, San Jose, CA, USA); Vimentin (Thermo Fisher Scientific, Fremont, CA, USA); and Snail, Notch2, HSPA1L, ALDH1A1, ALDH1A3 (Abcam, Cambridge, UK), and Oct4 (Millipore, Billerica, MA, USA) were used. Protein concentrations were determined using a Lowry kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on 8% or 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose membrane (Hybond; Amersham Pharmacia). The blots were blocked for 1 h at room temperature with blocking buffer (10% nonfat milk in PBS containing 0.1% Tween 20). The membrane was incubated overnight in a cold chamber with specific antibodies. After being washed with TBS, blots were developed with a peroxidase conjugated secondary antibody, and proteins were visualized via enhanced chemiluminescence procedures (Amersham) following the manufacturer’s protocol.