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REAGENTS/MATERIAL:
Immunoblotting
The following primary antibodies were used:
monoclonal anti-α-Syn, it recognizes total α-Syn including phosphorylated and non-phosphorylated forms (42) (BD Transduction Laboratories, Franklin Lakes, NJ, USA);
monoclonal anti-Ser129-phosphorylated α-Syn (psyn 64) (Wako, Osaka, Japan);
monoclonal anti-β-actin (AC-15) (Sigma);
polyclonal rabbit anti-GRK2 (C15) (Santa Cruz Biotechnology, Santa Cruz, CA, USA);
polyclonal rabbit anti-GRK3 (C14) (Santa Cruz Biotechnology);
monoclonal hamster anti-GRK5 against amino acid residues 94–157 of GRK5 (139) (Dr. M Sakamoto);
polyclonal rabbit anti-GRK6 (C20) (Santa Cruz Biotechnology);
polyclonal goatanti-casein kinase (CK) 2α’ (C20) (Santa Cruz Biotechnology)
and polyclonal rabbit anti-ubiquitin (1:1,000, Dako, Glostrup, Denmark).
Pulse-chase metabolic labeling experiments: After harvesting cells,
immunoprecipitation using Syn-1 antibody or anti-Ser129-phosphorylated α-Syn antibody was
carried out.
Immunocytochemistry: Wt-aS/SH cells
were labeled with monoclonal anti-α-Syn (LB509) (Covance, Emeryville, CA, USA)
and anti-ubiquitin polyclonal antibody.
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