Immobilon polyvinylidene difluoride transfer membranes

Thirty micrograms of different protein fractions from rat livers were subjected to 10% SDS-polyacrylamide gel electrophoresis, as described previously [11] . Briefly, proteins were transferred to Immobilon polyvinylidene difluoride transfer membranes (Millipore, Bedford, MA, USA) and blocked for 1 hr at room temperature with 5% non-fat milk solution in TBS-0.1% Tween-20. Detection was achieved using the enhanced chemiluminescence (ECL) kit for horseradish peroxidase (HRP) (Amersham Biosciences). To confirm the uniformity of protein loading, the blots were stained with -actin (Sigma-Aldrich) as a control. The size of detected proteins was estimated using protein molecular-mass standards (Invitrogen, Life Technologies). All antibodies were obtained from Santa Cruz Technologies, except those for insulin receptor substrate-2 (IRS-2) and phospho-protein kinase B (p-Akt), which were obtained from Cell Signaling (Danvers, MA, USA).

Thirty micrograms of different protein fractions from rat tissues were subjected to 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon polyvinylidene difluoride transfer membranes (Millipore, Billerica, MA, USA) and blocked for 1 h at room temperature with 5% non-fat milk solution in 0.1% Tween-20-Tris-buffered saline (TBS). Membranes were then incubated overnight with the primary antibody raised against Nrf2 or HO-1, in 0.1% Tween-20-TBS with 5% bovine serum albumin (BSA) at 4ºC.
Detection was achieved using the enhanced chemiluminescence (ECL) kit for horseradish peroxidase (HRP) (Amersham GE Healthcare Europe GmbH, Barcelona, Spain). To confirm the uniformity of protein loading, the blots were incubated with β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA) as a control. The size of detected proteins was estimated using protein molecular-mass standards (Invitrogen, Life Technologies). Primary antibodies for HO-1 and Nrf2 were obtained from Santa Cruz Biotechnologies (Dallas, TX, USA).

Thirty micrograms of different protein fractions from rat livers were subjected to 10% 10 SDS-polyacrylamide gel electrophoresis, as described previously [5] . Briefly, proteins were 11 transferred to Immobilon polyvinylidene difluoride transfer membranes (Millipore, Bedford, 12 MA, USA) and blocked for 1 hr at room temperature with 5% non-fat milk solution in TBS-13 0.1% Tween-20. Detection was achieved using the enhanced chemiluminescence (ECL) kit 14 for horseradish peroxidase (HRP) (Amersham Biosciences). To confirm the uniformity of 15 protein loading, the blots were stained with -actin (Sigma-Aldrich) as a control. The size of 16 detected proteins was estimated using protein molecular-mass standards (Invitrogen, Life 17 Technologies). All antibodies were obtained from Santa Cruz Technologies, except that for 18 insulin receptor substrate-2 (IRS-2) which was obtained from Cell Signaling (Danvers, MA, 19 USA). 20 21

Different protein extracts (20-30 µg) from rat frontal cortex and hippocampus were subjected to SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to Immobilon polyvinylidene difluoride Transfer Membranes (Millipore, Billerica, MA, USA), blocked for 1 h at room temperature with 5% non-fat milk solution in Tris-buffered saline (TBS) with 0.1% Tween-20, and incubated as described previously [21] (link). Detection was performed using the ECL chemiluminescence kit for HRP (Amersham GE Healthcare Europe GmbH, Barcelona, Spain).
To confirm the uniformity of protein loading, blots were incubated with -actin antibody (Sigma-Aldrich, St. Louis, MO, USA), which was used as the normalization control for Western blot analysis. Primary antibodies for total and phosphorylated Akt, and total and phosphorylated GSK3β were supplied by Cell Signaling (Danvers, MA, USA), and those for Drp1, IDE, Mfn2 and PSD95 were obtained from Abcam (Cambridge, UK). The anti-SYP antibody was obtained from Dako (Agilent Technologies, Santa Clara, CA, USA), Opa1 from BD Biosciences (San Jose, CA, USA), total Tau from Chemicon International (Temecula, CA, USA) and phosphorylated Tau from Invitrogen (Carlsbad, CA, USA).