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Primary antibodies diluted in blocking solution were then added and incubated overnight at 4 °C.

Membranes were then washed with blocking solution, and appropriate secondary antibodies diluted in blocking solution were then applied for 1 h at room temperature.

The primary antibodies (against biotin and the protein partner to be tested) diluted in blocking solution were then added, and the mixture was incubated overnight at 4 °C.

The respective HRP (horseradish peroxidase -conjugated anti-IgG secondary antibodies (1:2000 for N- and C- terminal NDRG1, 1:1000 for peroxidase -conjugatedl diluted in blocking solution) were then incubated for 1 h at room temperature.

The anti-Sirt1 antibody in blocking solution was then added (1∶1,000) and incubated overnight at 4°C.

A volume of 100 μl of blocking solution was then added to each well and gently mixed for 30 seconds.

Monovalent Fab fragments of goat anti-human IgM+IgG (Jackson Immunoresearch Laboratories, Inc ., in blocking solution, was then applied for secondary blocking.

The blocking solution was then discarded and 100 μl A2L2 cells (2 × 10-1 × 10) was added to each well of the ELISPOT plate.

Alkaline Phosphatase-conjugated anti-digoxigenin antibody (Roche Biochemicals, Basel, Switzerland) in blocking solution was then added to slides and incubated overnight at 4°C in a humidified chamber.

Incubation with primary antibodies diluted in blocking solution was then carried out for 1 hr at room temperatures or overnight at 4°C.

Secondary antibody (rabbit anti-sheep HRP diluted to 200 μg·mL−1 in block or sheep anti-mouse HRP diluted to 400 μg·mL−1 in blocking solution) was then incubated for 60 min and subsequently washed four times in PBS.