Phospho mapk array

COLO205 and SW620 colorectal cancer cells were transfected with 100 nM control siRNA or TESC siRNA and subjected to survival analysis using the WST-1 assay (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's protocol. Relative levels of phosphorylated mitogen-activated protein kinases (MAPKs) and other serine/threonine kinases were determined using a phospho-MAPK array from R&D systems, Inc. (Minneapolis, MN) according to the manufacturer's protocol [41 (link)].

Protein lysates from cells and tissue were collected and lysed in RIPA buffer with Halt Protease inhibitor cocktail (Thermo Scientific, #1861278), and phosphatase inhibitor cocktails 2 and 3 (Sigma, P5726 and P0044)^{72 (link)}. Protein concentration was determined by DC protein assay (Bio-Rad, #500). For proteome profiling, samples were analyzed according to manufacturer instructions using the Phospho-MAPK Array (R&D Systems, #893909). For western blots, 30 μg protein/condition were separated by SDS–PAGE (Thermo Scientific, NW0412A) and transferred to nitrocellulose membrane. Membranes were blocked with 5% milk and probed with 1:1000 Sprouty2 (Sigma, #AV50523) overnight at 4 °C, 1:1000 total and phospho-GSK3beta (S9; Cell Signaling, #12456 and #5558, respectively), 1:1000 total and phospho-Akt (S473; Cell Signaling, #2920 and #4060, respectively), or 1:10,000 mouse anti-Actin (Sigma, A1978) for 1 h at room temperature, followed by 1:20,000 IRDye-conjugated donkey anti-rabbit (LI-COR, #926-68023) and donkey anti-mouse (LI-COR, #926-32212) for 1 h at room temperature and quantification on an Odyssey imager (LI-COR) with Image Studio 4.0. Original scans are included in the source data file.

For an initial screening of the phosphorylation status of a variety of kinases after BCR and/or TLR9 activation, we have performed a phospho-MAPK array (R&D Systems, Minneapolis, MN, USA). 3×10⁶ BJAB cells were treated with various stimuli per sample for 30 minutes at 37°C. The experiment was carried out according to the manufacturer's instructions.