Sb202190

Sertraline hydrochloride, U0126, and SB202190 were purchased from Wako Pure Chemicals Industries (Osaka, Japan). Fluoxetine hydrochloride was purchased from LKT Labs (St. Paul, MN, United States). Imipramine hydrochloride and mianserin hydrochloride were purchased from Sigma–Aldrich. Fluvoxamine maleate and SP600125 were purchased from Tokyo Chemical Industries (Tokyo, Japan). Other common laboratory reagents were also obtained from commercial sources.

Recombinant soluble human TGF-β1 and TWEAK were from Peprotech (Rocky Hill, NJ, USA). Recombinant soluble human TNF-α was obtained from eBioscience (San Diego, CA, USA). Purified anti-α-tublin and anti-human Vimentin (V9) monoclonal antibodies (mAbs) SB431542 and AG1478 were from Sigma Chemicals (St. Louis, MO, USA). Anti-human E-cadherin (HECD-1) was from Takara (Tokyo, Japan). N-cadherin and anti-EGFR mAbs were from BD Biosciences (San Jose, CA, USA). Anti-phospho-EGFR (pY845) mAbs was from abcam (Cambridge, UK). Anti-Smad2/3, anti-phospho-Smad2 (Ser465/467), anti-extracellular signal-regulated kinase (ERK), anti-phospho-ERK (Thr202/Tyr204), anti-p38 MAPK, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-Akt, anti-phospho-NF-κB p65 (Ser536) polyclonal antibodies, and anti-ZO-1, anti- Jun N-terminal kinase (JNK), anti-phospho-JNK (Thr183/Tyr185), anti-phospho-Akt (Ser473), and anti-NF-κB mAbs were obtained from Cell Signaling Technology (Beverly, MA, USA). SB202190, SP600125, LY294002, and BAY11-7082 were from Wako Chemicals (Osaka, Japan). AZD6244 was from Selleckchem (Houston, TX, USA). Bronchial epithelial growth medium (BEGM) was purchased from Cambrex (East Rutherford, NJ, USA).

The p38 MAPK inhibitor SB202190 HCl and the SAPK/JNK inhibitor SP600125 were purchased from Wako Pure Chemical Industries Inc. KO‐MEFs were cultured in the complete DMEM medium for 20 h, and then SB202190 (3 μm final) or SP600125 (25 μm final) was added to the culture medium. After a 1‐h incubation with the inhibitor, the culture medium was changed to EMEM containing 1% FBS in either the presence or absence of the inhibitor. After 24 h in the new medium, cell lysates were prepared, and protein expression was analyzed using western blot analysis as described 6. Total RNA was extracted after a 6‐h incubation under the culture conditions mentioned above, cDNA was synthesized, and p21 mRNA was measured by qPCR as described 6. Effects of SB202190 on cell growth were measured after 48 h in the culture conditions mentioned above. The number of viable cells in each treatment was determined using trypan blue exclusion.
KO‐MEFs were cultured in the complete DMEM medium for 20 h, and then cells were incubated with the complete DMEM medium containing 0.1, 1, or 2 μm of MG132 (Merck) for 1 h prior to l‐Ser depletion with the same concentration of MG132 in EMEM containing 1% FBS for 6 h. Cell lysates were then prepared, and Ccnd1, phospho‐Rb, and Rb were analyzed by western blotting as described above.