Rabbit anti phospho mtor ser2448

The muscle supernatants were subjected to Western blotting as described previously [31 (link)]. Phosphorylation of mTOR at Ser2448 was detected using rabbit anti-phospho-mTOR (Ser2448) (Cell Signaling Technology, Danvers, MA, USA) and expressed as the ratio of total mTOR expression, determined using anti-mTOR (Cell Signaling Technology, Danvers, MA, USA). Phosphorylation of Akt at (Ser473) was detected using rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA) and expressed as a ratio of total Akt expression, determined using anti-Akt (Cell Signaling Technology, Danvers, MA, USA).

For Western blotting and immunofluorescence, the following primary antibodies were used: rabbit anti-LC3 [PM036] (1:100 for immunofluorescence; 1:500 for Western blot), rabbit anti-phospho-p62 [PM074] (Ser351) (1:500) from Medical & Biological Laboratories (MBL); rabbit anti-mTOR [#2972] (1:1000), rabbit anti-phospho-mTOR (Ser2448) [#2971] (1:100 for immunofluorescence; 1:1000 for Western blot), rabbit anti-p70S6 kinase [#2708] (1:1000), and rabbit anti-phospho-p70S6 kinase (Thr389) [#2708] (1:1000) from Cell Signaling; guinea pig anti-p62 [P0G-GP62-C] (1:1000) from Progen; rabbit anti-ATP6V0A4 [ab97440] (1:1000) from AbCam; mouse anti-β-tubulin [T4029] (1:1000) and mouse anti-β-actin [A5316] (1:1000) from SIGMA; and rabbit Spin1/SPNS1 (1:100) from original antibodies^{12 (link)}.

Total brain tissues were sonicated using a LabSonic homogenizer (B. Braun Biotech Inc., Allentown, PA, USA), and the protein concentration in the brain samples was then quantified using a bicinchoninic acid assay kit (Pierce, CA, USA). Samples were then analyzed by 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 5% milk containing 0.05% Tween in PBS. The blots were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:3000; Sigma-Aldrich), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-GRP78, mouse anti-CHOP, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308), rabbit anti-Akt, rabbit anti-phospho-4E-BP1, rabbit anti-phospho-p70 S6 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel). Membranes were then probed with secondary horseradish peroxidase-conjugated antibodies (1:3000; Amersham Biosciences, Little Chalfont, UK) for 1 h at room temperature. The blots were then visualized using chemiluminescent peroxidase substrate (ECL prime; GE Healthcare). Immunoreactivity for each protein band intensity was quantified using NIH ImageJ software [24 (link)] and normalized to β-actin as a loading control.