TLR2 activates the NFκB, PI3K/Akt, and ERK pathways in Meg-01 cells. (A) Meg-01 cells were treated for up to 1 hour with 1μg/mL of Pam3CSK4 (PAM). Western blots were run with samples at each time point and probed for phospho-NFκBp65, total NFκBp65, IκB, phospho-Akt, total Akt, phospho-ERK, and total ERK. Western blots are representative of 3 independent experiments, and results were compared with NT. (B) Meg-01 cells were pretreated for 1 hour with 25 μg/mL of IgG, anti-TLR2, or anti-TLR1 antibodies. Cells were then treated for 30 minutes with 1μg/mL of Pam3CSK4. Western blots are representative of 4 experiments. (C) Isolated megakaryocytes from WT and TLR2^−/− mice that were treated for 30 minutes with 1 μg/mL of Pam3CSK4 were run on Western blots and probed for phospho-NFκBp65 and total NFκBp65. Western blots are representative of 3 independent experiments. (D) Meg-01 cells were treated for up to 1 hour with 1 μg/mL of Pam3CSK4 (PAM). Western blots were probed for GATA-1, NF-E2, phospho-mTOR, total mTOR, and actin (loading control). Western blots are representative of 4 independent experiments.