Figure 5

JNK prevents Cdc20-mediated degradation of SMAR1. (a) JNK regulates the stability of SMAR1. MCF7 cells were treated with increasing concentrations of JNK inhibitor for 12 h. Cell lysates were immunoblotted for indicated proteins. (b) JNK prevents proteasomal degradation of SMAR1. MCF7 cells were either untreated or treated with 30 nM JNK inhibitor in the absence and presence of 10 μM MG132. JNK treatment was for 12 h and MG132 was for last 6 h. Cell lysates were immunoblotted for indicated proteins. (c) Relative mRNA level of SMAR1 upon JNK inhibition.SMAR1 mRNA levels were measured using RT-qPCR assay and normalized against GAPDH mRNA. NS represents not significant difference by one way ANOVA test. (d) SMAR1 interacts with JNK. Endogenous SMAR1 was immunoprecipitated and immunoprecipitates were immunoblotted with indicated antibodies. (e) JNK inhibition leads to reduced level of phospho serine of SMAR1. Endogenous SMAR1 was immunoprecipitated and immunoprecipitates were immunoblotted with indicated antibodies. (f) JNK inhibition leads to enhancement of K48-linked polyubiquitylation of SMAR1. MCF7 cells were treated with or without 30 nM JNK inhibitor and cell lysates were immunoprecipitated for SMAR1.Immunoprecipitates were immunoblotted with K48-linkage specific ubiquitin antibody. (g) Cdc20 degrades SMAR1 upon JNK inhibition. NS and Cdcd20KD cells were treated with JNK inhibitor, cells were lysed and immunoblotted for indicated proteins