1. A SP
N1-methyl
Con SP
116 kDa
IP GluR2L,
blot T912(P)
GluR2L
116 kDa
IP GluR2L,
blot GluR2L
116 kDa
IP GluR4,
blot T855(P)
GluR4
116 kDa
IP GluR4,
blot GluR4 B
PP->AA
TAT-JBD
Con
TAT-JBD
116 kDa
IP GluR2L,
blot T912(P)
GluR2L
116 kDa
IP GluR2L,
blot GluR2L
116 kDa
IP GluR4,
blot T855(P)
GluR4
116 kDa
IP GluR4,
blot GluR4
Supplementary Figure 3: Inactive analogs of JNK inhibitors do not affect GluR2L-Thr912 or GluR4-Thr855 phosphorylation. A: Cortical cultures (DIV17-21) were incubated for 1 h in aCSF containing DMSO vehicle control, with 20 mM SP or with 20 mM N1-methyl derivative of SP that inhibits JNK >20-fold less potently than SP itself. GluR2L and GluR4 were immunoprecipitated from lysates. Immunoprecipitates were subjected to SDS-PAGE prior to immunoblotting with the indicated antibodies. B:Peptides containing
wild type and mutant (Arg156, Pro157 -> AlaAla; residues critical for JNK interaction - see Barr et al., 2002) JBD sequences fused to the HIV-1 TAT sequence were added to primary cortical neuronal cultures (final concentration 30 mM) for 1h. GluR2L and GluR4 were immuno-precipitated from lysates. Immunoprecipitates were subjected to SDS-PAGE prior to blotting with the indicated antibodies.
Reference: Barr RK, Kendrick TS, Bogoyevitch MA (2002) Identification of the critical features of a small peptide inhibitor of JNK activity. J Biol Chem 277,