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To keep the scanning procedure within time limits, only 11 of the 22 sentences per block were probed in this way.
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Phospho-IGF-1Rβ (Tyr1131) and phospho-ERK1/2 (Thr202/Tyr204) were probed in 5% BSA-TBST buffer, whereas phospho-IGF-1Rβ (Tyr1135/Tyr1136) and phospho-Akt (Ser473) were probed in I-Block buffer.
Blocked membranes were probed with rabbit anti-mouse Nrf2 (1:5000) in 10 ml of 2% bovine serum albumin (BSA) for 1 h.
After blocking the membranes with blocking buffer, the membranes were probed with antibodies in recommended concentration as described in usage instruction antibodies.
After blocking, the membranes were probed with the keratin 6a primary antibody (1 500) (Abcam) in blocking buffer overnight.
Nonspecific binding was blocked by 1 hour incubation with blocking buffer before membranes were probed overnight at 4°C with primary antibodies diluted in blocking buffer (3% BSA in Tris-buffered saline with 0.1%Tween-20Tween-20)).
Membranes were probed with antibodies in the blocking solution for an additional hour.
After blocking in non-fat dried milk, membranes were probed with primary antibody in a solution of PBS containing 0.1% Tween20.
After three washes in TBST, membranes were probed with the indicated antibodies in blocking buffer overnight.
After blocking with 0.5% blocking reagent (Roche Diagnostics), filters were probed with specific antibodies.
Membranes were probed with primary antibody in blocking buffer at 4°C overnight.
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