Horseradish peroxidase hpr conjugated secondary antibody

The general steps of the Western blot procedure have been described previously [49 ]. Briefly, after treatment with the plant extracts VSM and BORM for at least 48 h the cells were trypsinized, washed with PBS and lysed in ice-cold lysis buffer (Bio-Plex Cell Lysis Kit, Bio-Rad, USA). After SDS-PAGE, protein content per lane as well separation quality was controlled with the Criterion Stain FreeTM gel imaging system (Bio-Rad, Germany). For protein detection primary antibodies (PCNA: sc-56, from Santa Cruz, USA; BCL-2: B3170, from Sigma) were incubated overnight at 4 °C followed by labeling with a horseradish peroxidase (HPR)-conjugated secondary antibody (Dako, Glostrup, Denmark) for 1 h at room temperature. Protein signals were visualized by using SuperSignal West Femto Chemiluminescent Substrate (Pierce Biotechnology, Rockford, USA). Band intensity was analyzed densitometrically with the Molecular Imager ChemiDoc XRS and Image Lab 3.0.1 software (Bio-Rad, USA). Protein detection was repeated at least three times with individually prepared cell lysates from independently passaged cells.

Western blotting procedure was performed as already described^{22 (link)}. For protein detection, primary antibodies anti-GSCH (#16726-1-AP; Proteintech Europe, Manchester, UK), anti-GAPDH (#D16H11; Cell Signaling, USA), anit-PCNA (#sc-56; Santa Cruz, USA); anti-β-Actin (#4970; Cell Signaling, USA), anti-AMT (#sc-99267; Santa Cruz, USA), anti-cSHMT (#sc-365203; Santa Cruz, USA), anti-mSHMT (#sc-390641; Santa Cruz, USA), anti-GNMT (#sc-166834; Santa Cruz, USA) were incubated overnight at 4 °C followed by labeling with a horseradish peroxidase (HPR)-conjugated secondary antibody (Dako, Glostrup, Denmark) for 1 h at room temperature. Band intensity was analyzed densitometrically with the Molecular Imager ChemiDoc XRS and Image Lab 3.0.1 software (Bio-Rad, München, Germany).