Western blot analysis
Western blot analysis of hippocampus samples from healthy subjects and AD patients.
Steps to reproduce
Brain samples were homogenized (100 µL for 10 mg of tissue) in 20 mM Hepes pH 8.0, 1 mM EGTA, 0.4% NP-40, and added of complete protease- (11836153001, Roche) and phosphatase-inhibitor (5870, Cell Signaling) cocktails following the manufacturer’s instructions. Brain samples were homogenized using a pellet micro-pestle and then subjected to sonication (3 cycles, 20 s each). The lysates were centrifuged at 13000 g for 10 min at 4 °C and, after discarding the pellet, the concentration of total proteins in the supernatant was determined using the Bradford reagent (500-0205, BioRad). For each sample 40 µg of total proteins were subjected to SDS-PAGE and transferred on a PVDF membrane using the Mini Trans-Blot Cell system (BioRad). The membranes were developed using rabbit anti-PHGDH (HPA024031, Sigma, dilution 1:1000), anti-PSAT (abin2856767, Antibodies online, 1:1000) or anti-PSP (PA5-22003, Invitrogen, dilution 1:1000) antibodies. The membrane was blocked overnight at 4 °C with 4% dried milk in Tris-saline buffer pH 8.0 added of 0.1% Tween 20 and subsequently incubated with primary antibodies diluted in 2% dried milk in Tris-saline buffer pH 8.0 added of 0.05% Tween 20 for 2 h at room temperature. After extensive washing, the membrane was incubated for 1 h at room temperature with rabbit IgG (Alexa-Fluor Plus 800, 1:20000 dilution in Tris-saline buffer pH 8.0 added of 0.05% Tween 20). Western blots were analyzed by Li-cor ImageStudio software: the intensity signal of each sample was normalized by the GAPDH signal (detected using a mouse anti-GAPDH, 1:2000, MA5-15738 Invitrogen and a mouse IgG IRDye 680 1:5000). The content of each protein was calculated by the software based on the intensity of known amounts of recombinant proteins and was related to the g of total proteins loaded into the gel. Controls included the addition of a known amount of recombinant proteins to the samples. Each sample was analyzed at least three times (in three different SDS-PAGE runs). The results were analyzed using Prism (Graphpad Software Inc.). The D’Agostino and Pearson normality test was used to analyze the distribution of variables: since a non-normal distribution was observed, a non-parametric approach was used for all statistical analysis. Variation of protein levels between controls and AD patients was evaluated using the two-tailed Mann-Withney test. A p value <0.05 was considered as statistically significant.