Hrp conjugated anti rabbit secondary antibody

1.5 × 10⁶ cells (2008-C13) were plated in 100 mm cell culture dish and allowed to attach overnight. After 48 hours, cells were lysed with ice-cold lysis buffer supplemented with the protease inhibitor cocktails (Roche Molecular Biochemicals, Mannheim, Germany). The protein content was determined by Lowry procedure (Bio-rad DC Protein Assay, MA, USA). Equal amounts of protein (40 μg) were loaded on a polyacrylamide gel and electrophoretically separated in running buffer. After electrophoresis, the proteins were blotted onto an Hybond-P PVDF membrane (Amersham Biosciences, Buckinghamshire, UK). After blocking, the membrane was exposed to the elected primary antibodies: anti-LC3 (1:1000; Cell Signaling, MA, USA) or anti-G6PD (1:500; Santa Cruz Biotechnology, Inc., Europe). After washing, the membrane was incubated with HRP-conjugated anti-rabbit secondary antibody (1:3500; PerkinElmer, MA, USA). The signal was visualized with enhanced chemoluminescent kit (Amersham Biosciences) according to the manufacturer's instructions and analyzed by Molecular Imager VersaDoc MP 4000 (Bio-rad). LC3 and G6PD were normalized to beta-actin (1:7000; AbCam, Cambridge, UK).

The Lowry method was used to evaluate the protein content after the cells had been seeded in 6-well plates, cultured in complete media, and lysed using ice-cold lysis buffer supplemented with protease inhibitor cocktails (Roche Molecular Biochemicals, Mannheim, Germany) (Biorad DC Protein Assay, MA, USA). In the running buffer, 25 µg of protein from each sample was placed onto a polyacrylamide gel and electrophoretically separated. The proteins were blotted onto a Hybond-P PVDF membrane following electrophoresis (Amersham Biosciences, Buckinghamshire, UK). The membrane was exposed to anti-TOM20 (mouse, 1:1000; AbCam, Cambridge, UK), anti-VDAC1 (mouse, 1:1000; AbCam, Cambridge, UK), and anti-BNIP3 (rabbit, 1:1000; AbCam, Cambridge, UK) after blocking with a 10% skim milk solution. Following washing, the membrane was incubated with HRP-conjugated anti-rabbit secondary antibody (1:3500; PerkinElmer, MA, SUA) or anti-mouse secondary antibody (1:10000; PerkinElmer, MA, USA). According to the manufacturer’s instructions, the signal was seen using an improved chemiluminescent kit from Amersham Biosciences, and then it was examined using a Molecular Imager VersaDoc MP 4000 (Biorad, Hercules, CA, USA). Proteins were normalized to β-ACTIN (mouse, 1:7000, AbCam, Cambridge, UK) and GAPDH (rabbit, 1:2000, Cell Signaling, Danvers, MA, USA).

6-multiwell plates were seeded with 75x10³ cells and incubated overnight. Cells were then treated according to the protocol and then lysed with ice-cold lysis buffer, supplemented with the protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany). Cell lysates were then centrifuged at 14000 rpm for 15 minutes at 4° C and the supernatant protein content was determined by the Lowry procedure (Bio-Rad DC Protein Assay, MA, USA).
Equal amount of protein was loaded on a polyacrylamide gel and electrophoretically separated in running buffer. After electrophoresis, the proteins were blotted onto a Hybond-P PVDF membrane (Amersham Biosciences, Buckinghamshire, UK). After blocking with a 10% skim milk solution, the membrane was exposed to the primary antibody anti-SIRT1 (1:250; Sigma-Aldrich, St Louis, MO, USA), and, following overnight incubation, was washed and exposed to HRP-conjugated anti-rabbit secondary antibody (1:3500; PerkinElmer, MA, USA). The signal was visualized with an enhanced chemoluminescent kit (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions and analyzed by Molecular Imager VersaDoc MP 4000 (Bio-Rad, Hercules, CA, USA). All the proteins were normalized to calnexin (1:1000; Santa Cruz Biotechnology INC, Santa Cruz, CA, USA).