Anti gadd153

The relevant procedures have been described in a previous report [8 (link)]. Proteins were reacted with one of the following: anti-LC3A (#4599), anti-LC3B (#3868), anti-PARP (#5625), anti-GADD153 (#2895), anti-GRP78 (#3177), anti-t-Akt (#9272), anti-p-Akt Ser473 (#9271), anti-p-ERK (#4370), anti-p-p70S6K Thr389 (#9234) purchased from Cell Signaling (Danvers, MA), anti-p62 (GTX100685) obtained from GeneTex (Irvine, CA) or monoclonal anti-β-actin (Sigma, AC-40). Immunohistochemistry (IHC) was carried out with polyclonal anti P-gp, followed by anti-goat IgG (Santa Cruz Biotechnology) conjugated to horseradish peroxidase. Hematoxylin was used for counterstaining.

Longistyline C was isolated from the leaves of pigeonpea by our laboratory [18 ] and the purity is over 95% which was detected by high performance liquid chromatography (HPLC) method.
Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY), penicillin (100 IU/ml Sigma, St. Louis, MO), streptomycin (10 μg/ml; Sigma), 10% heat-inactivated fetal calf serum (Gibco, Grand Island, NY). PC12 cells were purchased from the Cell Bank of Peking Union Medical College (Beijing, China). Methyl thiazolyl tetrazolium (MTT), dimethylsulfoxide (DMSO) and glutamate were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA), Fura-2/AM (Beyotime Institute of Biotechnology, Jingsu, China). Antibodies used for western blot analysis, primary antibodies: 1:1000 Anti-GADPH, 1:200 Anti-ERK1/2, 1:200 Anti-β-Actin, 1:100 Anti-GRP78, 1:200 Anti-p-ERk1/2,1:250 Anti-Caspase-12, 1:100 Anti-GADD153, 1:200 Anti-XBP-1, 1:500 Anti-Caspase-9, 1:1000 Anti-NMDAR/NR2B, 1:1000 Anti-CaMKII, 1:1000 Anti-CREB and 1:1000 Anti-p-CREB (Cell signaling Technology, USA); secondary antibodies: 1:1000 Anti-mouse-HRP and 1:1000 Anti-rabbit-HRP (Santa Cruz biotechnology, USA); Nonfat milk powder and bovine serum albumin (Becton, Dickinson and Company, USA). Double distilled water was used in all experiments. All the reagents were of analytical grade.