Goat anti mouse horseradish peroxidase hrp conjugated secondary antibody

Cells were grown to ~70% confluency in DMEM including 10% Foetal Bovine Serum. The IP was carried out with rabbit polyclonal anti-MTDH (Abnova (H00092140-PW2), Dallas TX, USA) and performed as described previously^{59 (link)}. MTDH IP was valuated using Western blotting analysis, according to standard procedures. Briefly, gel electrophoresis and protein transfer were performed using XCell SureLock Mini-Cell with Blot Module™ (Invitrogen, Life Technologies). Membranes were blocked and incubated for at least 16 hours at room temperature with mouse polyclonal anti-MTHD (Abnova (H00092140-PW2), Dallas TX, USA; dilution 1:500). Following incubation with goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Dako, Glostrup, DK; dilution 1:10.000) proteins were detected using ECL prime Western Blotting Reagent (GE Healthcare, Chalfont St. Giles, UK) and ChemiDoc-It Imaging System (UVP, Upland, CA, USA). The β-actin was visualized using HRP-conjugated mouse anti-β-actin (Abcam; dilution 1:10.000).

Protein extracts were prepare as described (Link et al., 2006 (link)). TgfβR2 protein was detected by a primary anti-tgfβR2 antibody (diluted 1:250 in blocking solution, sc-17792; Santa Cruz Biotechnology) followed by a goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000 in blocking solution, P044701-2, DAKO). An anti β-actin-HRP-conjugated antibody (1:35,000, A3854; Sigma) was used for loading control.