Immunoblotting was performed as described earlier (Siddiqi et al., 2006b (link)). In brief, protein samples containing equal amount of protein were separated by SDS-PAGE, transferred on to a nitrocellulose membrane (Bio-Rad, Hercules, CA) and the membrane was blocked with 10% (w/v) non-fat dried skimmed milk in PBS-T. The membrane was incubated with a specific primary (as indicated in figures) and secondary antibodies conjugated with HRP. Protein detection was performed with ECL reagents using autoradiography film (MIDSCI, St. Louis, MO).
For co-immunoprecipitation analysis, protein samples (250 μg prot) were solubilized in 2% triton X-100 in PBS followed by incubation with indicated primary antibodies for 4 hours at 4°C. Post-incubation, secondary antibodies attached to agarose beads were added and incubated for 12–14 hours at 4°C. The resulting protein complexes attached to agarose beads were washed thoroughly and co-immunoprecipitated proteins were separated by SDS-PAGE as described previously (Siddiqi et al., 2010a (link); Tiwari et al., 2013 (link)).
For co-immunoprecipitation analysis, protein samples (250 μg prot) were solubilized in 2% triton X-100 in PBS followed by incubation with indicated primary antibodies for 4 hours at 4°C. Post-incubation, secondary antibodies attached to agarose beads were added and incubated for 12–14 hours at 4°C. The resulting protein complexes attached to agarose beads were washed thoroughly and co-immunoprecipitated proteins were separated by SDS-PAGE as described previously (Siddiqi et al., 2010a (link); Tiwari et al., 2013 (link)).
