Anti prongf

Protein extraction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection were performed as previously described.^{8 (link)} For immunodetection, antibodies against SIRT1 and the pro-peptide and active forms of caspase-3 and PARP, as well as the total and phosphorylated forms of Akt (Ser473; no. 4685), extracellular signal–regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204; no. 9101) and NF-κB p65 (Ser536; no. 3033), were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against TrkA (ab76291) and p75NTR (ab52987) were acquired from Abcam. Anti-proNGF was purchased from Alomone Labs (ANT-005). Anti-NGF (AB1528SP) and β-Actin (MAB1501) was acquired from Millipore (Temecula, CA, USA). For semiquantitative analyses, relative protein levels were expressed as fold induction compared with the negative control by normalizing the density of the protein of interest to an internal control.

Formalin-fixed and paraffin-embedded human and mouse livers were sectioned and used for hematoxylin and eosin and IHC staining, as previously described.^{8 (link)} Deparaffinized and rehydrated sections were treated for antigen retrieval and incubated with primary antibodies at 4 °C overnight. Rabbit monoclonal antibodies purchased from Abcam (clone E51, Cambridge, MA, USA) were used to localize NGF (ab52918), TrkA (ab76291), p75NTR (ab52987) and the cleaved peptides of poly(ADP-ribose) polymerase (PARP). Anti-proNGF was purchased from Alomone Labs (Jerusalem, Israel) (ANT-005). The antigen location in tissue sections was visualized with an horseradish peroxidase-linked polymer Envision detection system (DAKO, Glostrup, Denmark), followed by counterstaining with hematoxylin. Normal liver sections were treated with equimolar concentrations of isotype-matched normal IgG as negative controls.