Fig. 5

The ubiquitination of IRAK1 in TM4 cells after exposure to MBP. (a-c) The expression of IRAK1 in TM4 cells after exposure to different concentrations of MBP for 24 h. a The mRNA levels of IRAK1 were measured with q-PCR, and GAPDH was measured as a loading control. b, c The protein levels of IRAK1 were measured by Western blotting. The densitometry data were quantified with ImageJ (n = 3). GAPDH was assessed as an internal control. d MBP (0.1 mM) attenuates IRAK1 ubiquitination and stimulates K63-mediated IRAK1 polyubiquitination. Cell lysates were immunoprecipitated (IP) with anti-IRAK1, which was followed by Western blotting analysis with anti-K63 ubiquitin (K63-Ub), anti-ubiquitin (Ub), and anti-IRAK1 antibodies. e, f TM4 cells were pretreated with an IRAK1 inhibitor for 1 h, which was followed by 24 h treatment with 0.1 mM MBP. The expression levels of JNK and p-JNK were determined by Western blotting. The densitometry data were quantified with ImageJ (n = 3). GAPDH was assessed as an internal control. The results are expressed as the means ± SEM. ** p < 0.01; * p < 0.05. # p < 0.05, vs MBP exposure