Phospho erk1 erk2

Heart tissues were removed from liquid nitrogen, thawed, and weighed. Tissue homogenate was prepared with RIPA buffer (150 mM NaCl, 10% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, and 50 mM Tris base), supplemented with protease inhibitor cocktail (Sigma Aldrich, USA). Then homogenate was centrifuged at 12000 rpm for 15 min at 4°C. Protein concentration was measured by the method described by Bradford [20 (link)]. Each well was loaded with equal amount of proteins (40 μg) in a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membrane. Following this, membrane was probed with primary antibodies, that is, ERK1/ERK2 (Cell signaling, USA), phospho-ERK1/ERK2 (Thr202/Tyr204; Cell signaling, USA), SAPK/JNK (Cell signaling, USA), phospho SAPK/JNK (Thr183/Tyr185; Cell signaling, USA), p38 (Abcam; UK); phospho-p38 (Santa Cruz, USA), NFκBp65 (Cell signaling, USA), and β-actin (Cell signaling, USA) overnight at 4°C. These primary antibodies were detected by adding HRP-conjugated secondary antibodies (Merck Genei, India) after incubating for 2 h at room temperature. Later, the bound antibodies were visualized with an enhanced chemiluminescence (ECL) (Thermo Fischer Scientific Inc., USA) kit and quantified by densitometric analysis.

Oridonin (purity >99.5%) was purchased from Xi’an Haoxuan Biotechnique, China. It was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and stored at −20°C. Both N-acetyl-l-cysteine (NAC) and ATRA were purchased from Sigma-Aldrich. Recombinant human tumor necrosis factor (TNFα) was obtained from Peprotech (Rocky Hill, NJ, USA). Cycloheximide was purchased from Sigma-Aldrich. ERK inhibitor PD98059, p38 inhibitor SB203580, JNK inhibitor SP600125, and NF-κB inhibitor Bay 11–7082 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). When cells were treated with these reagents, matching concentrations of vehicle were used as the control and the final concentration of DMSO was kept at or below 0.1% in all experiments.
Antibodies recognizing p65, IκBα, and RARα were purchased from Santa Cruz Biotechnology. Antibodies recognizing phospho-IκBα (Ser32/Ser36), phospho-p65, IκB kinase beta (IKKβ), phospho-IKKα/β, phospho-ERK1/ERK2, ERK1/ERK2, phospho-p38, p38, phospho-JNK, and JNK were purchased from Cell Signaling Technology (Beverly, MA, USA).

Cell lysates were collected, and blotted as previously described [28 (link)]. c-Met (#8198), phospho-c-Met (Tyr1349; #3133), phospho-c-Met (Tyr1234/1235; #3077), Akt (#9272), phospho-Akt (Ser473; #9271), Erk1/Erk2 (#9107), phospho-Erk1/Erk2 (Thr202/204; #4376), EGFR (#2646), phospho-EGFR (Tyr1068; #3777), phosphor-EGFR (Tyr1173; #4407) ErbB3 (#4754), PARP (#9532) monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA). All antibodies were used at a 1:1000 dilution. -actin antibody (#A2228) was obtained from Sigma-Aldrich (St. Louis, MO) and was used at a 1:10,000 dilution.