MYD88 regulation of IRAK 1 and IκBα activity in WM cells-expressing the L265P mutation. (A) Western blot analyses were performed using antibodies which detect total and phosphorylated IRAK1 (The-209) and IκBα (Ser-32) following lentiviral transduction with either MYD88 knockdown (shRNA#1) or control lentiviral vectors. The relative density of phosphorylated vs total IRAK1 was analyzed by densitometry measurements and normalized to the values of control vectors. (B) To confirm the rapid degradation of activated IRAK1 that leading the reduction of total IRAK1 proteins, cells were treated with the proteasome inhibitor MG132 for 4 hours after MYD88 knockdown (shRNA#1). The phosphorylated and total IRAK1 were checked by western blot. (C) The effects of IRAK 1 and 4 inhibitor and MYD88 homodimerization inhibitory peptide were also evaluated by the phosphorylation of IRAK1 and IKBα with western blot. (D) Changes in phosphorylated IRAK1 in the presence of MG132 following treatment with either an IRAK 1 and 4 or MYD88 peptide inhibitor.