Anti phospho p44 42 mapk t202 y204

All the chemicals including kaempferol (purity: 95.0%) used in this experiment were purchased from Sigma (St. Louis, MO, USA) unless otherwise stated. Anti-NF-κB p65 (sc-8008), anti-specificity protein-1 (Sp1) (sc-17824), anti-inhibitory kappa Bα (IκBα) (sc-371), and anti-β-actin (sc-8432) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-nuclear matrix protein p84 (ab-487) antibody was purchased from abcam (Cambridge, MA, USA). Anti-phospho-EGFR (Y1068), phospho-specific anti-IκBα (serine 32/36, #9246), anti-EGFR, anti-phospho-IKKα/β (Ser176/180, #2687), anti-MEK1/2, anti-phospho-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2 (S221), anti-phospho-p38 MAPK (T180/Y182), anti-p38 MAPK, anti-phospho-p44/42 MAPK (T202/Y204), and anti-p44/42 MAPK antibodies were purchased from Cell Signaling Technology Inc (Danvers, MA, USA). Either Goat Anti-rabbit IgG (#401315) or Goat Anti-mouse IgG (#401215) was used as the secondary antibody and purchased from Calbiochem (Carlsbad, CA, USA).

293T cells with or without forced expression of EXOSC3 and CNOT4 were lysed with lysis buffer (10 mmol/L Tris‐HCl [pH 7.4], 5 mmol/L EDTA, 150 mmol/L NaCl, 10% glycerol, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS, 50 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride [PMSF], and 1 mmol/L sodium orthovanadate [Na₃VO₄]) and a protease inhibitor mixture (complete [EDTA‐free] protease inhibitor [Roche]) for 20 min on ice and centrifuged at 15,106 g for 15 min at 4°C. Supernatants were subjected to SDS‐PAGE, and the separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore). The membranes were incubated with primary antibodies at 4°C overnight and then incubated with horseradish peroxidase‐labeled secondary antibodies. The signals were developed using ECL™ Western Blotting Detection Reagents (GE Healthcare) and visualized with an LAS 4000 mini system (GE Healthcare). The antibodies used in this experiment were as follows: anti‐FLAG^® M2‐peroxidase (HRP) (Sigma), anti‐phospho‐p44/42 MAPK (T202/Y204) (D13.14.4E) XP™, anti‐phospho‐JNK, anti‐phospho‐STAT3 (Y705), anti‐phospho‐AKT (S473) (D9E) XP™ (Cell Signaling Technologies), anti‐MYD88 (Santa Cruz Biotechnology), and anti‐actin (clone C4) (Millipore).