Horseradish peroxidase hrp conjugated anti rabbit or anti mouse igg

For production of polyclonal antibodies, glutathione S-transferase (GST)-fusion proteins containing the specific antigenic regions of seven candidate proteins were expressed in Escherichia coli BL21 and affinity purified with glutathione Sepharose 4B (GE Healthcare). The recombinant proteins were used as antigens for producing rabbit polyclonal antisera. The antibodies were affinity purified using the appropriate proteins and an AminoLink immobilization kit (Pierce). The following commercially available antibodies were also used: a mouse monoclonal antibody against ADAM2 (1/1000, MAB19292) from Millipore; an antibody against α-tubulin (1/1000, T6199) from Sigma-Aldrich; and an antibody against GAPDH (1/1000, MCA4739) from Bio-Rad. As secondary antibodies for Western blot analysis, we used horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch).

Rabbit anti‐Npt2a and Npt2c polyclonal antibodies were generated as described previously and used for immunoblotting and immunohistochemistry (Ohkido et al., 2003; Segawa et al., 2005). Mouse anti‐actin monoclonal antibody (Millipore) was used as an internal control. Horseradish peroxidase (HRP)‐conjugated anti‐rabbit or anti‐mouse IgG was utilized as the secondary antibody (Jackson Immuno Research Laboratories, Inc, West Grove, PA, USA). The diluted antibodies for immunoblotting were as follows: anti‐Npt2a (1:15,000), ‐Npt2c (1:1,500), and ‐actin (1:10,000). The diluted antibodies for immunofluorescence staining were as follows: anti‐Npt2a (1:500), ‐Npt2c (1:1,000), and ‐villin (Millipore) (1:500).