Peroxidase conjugate anti mouse or anti rabbit igg

Immunoblotting was performed as previously described [27 (link)] using the following antibodies: anti-cyclin D1(DCS-6), anti-ATM (H-248), anti-DNA-PKcs (E-6), anti-H2AX (M-20), anti-p-H2AX (3C10), anti-RAD51 (3C10), anti-Ku86 (B-1), anti-p21waf1 (B-2), anti-p27Kip1 (A-10) and α-tubulin (B-7) all from Santa Cruz Biotechnology. Anti-pospho-Ser1981-ATM (10H11.E12), anti-pospho-Thr2609-DNA-PKcs (10B1) were from abcam^®. Peroxidase-conjugate anti-mouse or anti-rabbit IgG (Amersham-Pharmacia Biotech, UK or Santa Cruz) were used for enhanced chemiluminescence (ECL) detection.

Cells were lysed in 2% SDS containing 2 mM phenyl-methyl sulphonyl fluoride (PMSF) (Sigma), 10 μg/ml antipain, leupeptin and trypsin inhibitor, 10 mM sodium fluoride and 1 mM sodium orthovanadate (all from Sigma) and sonicated for 30 s. Proteins of whole-cell lysates were assessed using the Lowry method, and equal amounts were separated on SDS-PAGE. The proteins were transferred to a nitrocellulose membrane (Schleicher and Schuell, BioScience GmbH, Germany) by electroblotting. The balance of total protein levels was confirmed by staining the membranes with Ponceau S (Sigma). Immunoblotting was performed with the following antibodies: anti-β2-AR (H-20), anti-ERK2 (C-14, positive also for ERK1), anti-phospho-ERKs (E-4), anti-MEK 1/2 (9G3), anti-phospho MEK 1/2 (7E10), anti-GAPDH (6C5), Anti-β-actin (C4), and anti-Histone H3 (1G1) all from Santa Cruz Biotechnology (Santa Cruz, CA); anti-NRF2 (from Invitrogen #PA5-88084, Waltham, Massachusetts, USA). Peroxidase-conjugate anti-mouse or anti-rabbit IgG (Amersham-Pharmacia Biotech, UK, or Santa Cruz) were used for enhanced chemiluminescence (ECL) detection. Each western blot was performed in triplicate.

Cells were lysed in 2% SDS containing 2 mM phenyl-methyl sulphonyl fluoride (PMSF) (Sigma), 10 μg/ml antipain, leupeptin and trypsin inhibitor, 10 mM sodium fluoride and 1 mM sodium orthovanadate (all from Sigma) and sonicated for 30 s. Proteins of whole-cell lysates were assessed using the Lowry method and equal amounts were separated on SDS-PAGE. The proteins were transferred to a nitrocellulose membrane (Schleicher and Schuell, BioScience GmbH, Germany) by electroblotting. The balance of total protein levels was confirmed by staining the membranes with Ponceau S (Sigma). Immunoblottings were performed with the following antibodies: anti-ERK2 (C-14, positive also for ERK1), anti-phospho-ERKs (E-4), anti-phospho-Akt1 (5.Ser 473), anti-Akt1 (G5), anti-phospho-p38 (E1), anti-p38 (A1F7), anti-phospho-mTOR (59.Ser 2448), anti-mTOR (30), and α-tubulin (B-7) (all from Santa Cruz Biotechnology, Santa Cruz CA); anti-LC3B (from Abcam, Cambridge UK).
Peroxidase-conjugate anti-mouse or anti-rabbit IgG (Amersham-Pharmacia Biotech, UK, or Santa Cruz) were used for enhanced chemiluminescence (ECL) detection.