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However, the new state-of-the-art of signalling technologies permit a better bandwidth resulting in an exponential solution space.
Cells were lysed in 10× volume of M-PER lysis buffer (Pierce) + 100 mM NaF + 1 mM Na3VO4 + 1% protease inhibitors (Cell Signalling Technologies) + 150 mM NaCl + 1 mM EDTA at pH 8 and incubated for 10 min at room temperature.
Total lysates were subjected to Western blotting with phospho-S473 or phospho-T308 specific PKB antibodies (Cell Signalling Technologies).
Cells were processed for immunofluorescence using monoclonal 262K HA antibody (Cell Signalling Technologies) or polyclonal anti-UCHL1 (PGP 9.5, Biomol) as previously described [18].
Membranes were then glycine-stripped (25 mM glycine, pH 2.0, 1% SDS, 30 min at 22°C) and re-blotted with total PKB antibodies (Cell Signalling Technologies).
Antibodies against phospho Thr308-PKB (the PDK1 site) and pan-actin were purchased from Cell Signalling Technologies (Danvers, MA, USA) and NeoMarkers (Fremont, CA, USA), respectively.
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Network Rail said it was investing £350m to improve signalling technology.
Phospho-p38MAPK (Thr180/Tyr182) and p38MAPK were purchased from Cell Signalling Technology, Inc. (Milan, Italy).
Antibodies against SHP2 and p85 were from Cell Signalling Technology (UK).
pRb antibody-rabbit polyclonal; 1 1000, Cell Signaling Technologies: Ser807/811, FRα antibody-rabbit polyclonal; 1 500.
The lyophilized samples were resuspended in 1X IAP buffer (Cell Signaling Technologies).
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