Anti bip antibody

Proteins were separated on precast polyacrylamide gels (Bio-Rad) and transferred to Hybond (Amersham). For detection of α-synuclein, proteins were cross-linked to membranes using 0.4% paraformaldehyde (51 , 52 ). Chemiluminescent detection was performed with enhanced chemiluminescence (Amersham). Antibodies used were anti-α-synuclein antibody ([1:1500], BD Transduction Laboratories, 610787), anti-Ataxin-2 antibody ([1:4000], BD Biosciences, 611378), anti-Staufen antibody ([1:5000], Novus Biologicals, NBP1-33202), anti-BiP antibody ([1:3000], Genetex, GTX113340), anti-CHOP antibody ([1:3000], Cell Signaling Technology, 2895), anti-mTOR antibody ([1:4000], Cell Signaling Technology, 2972), anti-SQSTM1/p62 antibody ([1:4000], Cell Signaling Technology, 5114), anti-LC3B antibody ([1:7000], Novus Biologicals, NB100-2220SS), anti-tyrosine hydroxylase antibody ([1:2000], Millipore-Sigma, AB152), and peroxidase-conjugated monoclonal anti-β-Actin antibody (clone AC-15) ([1:20,000], Sigma-Aldrich, A3854). The secondary antibody was peroxidase-conjugated anti-rabbit (Jackson ImmunoResearch Laboratories).

DTT, dipyridyl, NH₄Cl, an anti-DYKDDDDK (FLAG) tag antibody, an anti-Myc tag antibody, leupeptin, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Diamide, N-ethylmaleimide (NEM), methoxypolyethylene glycol (PEG) maleimide, fetal bovine serum (FBS), penicillin-streptomycin solution, and S-methyl methanethiosulfonate (MMTS) were obtained from Sigma-Aldrich (St. Louis, MO). (N-[6-(biotinamido)hexyl]-3’-(2’-pyridyldithio) propionamide (Biotin-HPDP) and streptavidin-agarose were purchased from Pierce Biotechnology (Rockford IL). An anti-HSC70 antibody was purchased from Novus Biologicals (Littleton, CO). An anti-BiP antibody was purchased from GeneTex, Inc. (Irvine, CA). Dimethyloxaloylglycine (DMOG), Diamide, and a horseradish peroxidase (HRP) Maleimide Conjugate were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). KOD plus neo DNA polymerase was purchased from TOYOBO (Tokyo, Japan). Revert AidTM M-MuLV Reverse transcriptase was purchased from MBI Fermentas (Vilnius, Lithuania).