P p38 mapk thr180 tyr182

Phosphorylated and non-phosphorylated p38MAPK and ERK1/2 protein levels were determined by Western blot analysis as described previously [28 (link)]. The cell lysate was prepared with PhosphoSafe™ Extraction Reagent (Merck, Germany), and protein concentrations were determined with Bradford reagent (Bio-Rad Laboratories, USA). Proteins were separated, electrophoretically blotted onto nitrocellulose membranes, and washed with 10 mM Tris-HCl buffer (pH 7.6) containing 0.05% Tween-20; then, the membranes were incubated in blocking buffer (5% non-fat dry milk, 10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween-20) at room temperature for 3 h and with diluted primary antibodies (glyceraldehyde phosphate dehydrogenase [GAPDH 1:2000, pERK1/2 [Thr202/Tyr204] 1:1000, Santa Cruz Biotech, USA; p-p38MAPK [Thr180/Tyr182] 1:500, ERK1/2 1:1000, and p38MAPK 1:1000, Cell Signaling, USA) at 4 °C overnight. Bound antibodies were detected with HRP-conjugated anti-rabbit IgG (dilution 1:1000 and 1:8000) and visualized with a ChemiDocTM MP Imaging System (Bio-Lab, USA) and Image Lab 5.1 software (Bio-Lab, USA).

IK was isolated from the supercritical carbon dioxide (SC-CO₂) extract, a radiation-induced mutant cultivar of Perilla frutescens var. crispa [23 (link)]. Specific antibodies for p44/42 MAPK (ERK1/2), phospho (p)-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204), SPK/JNK, p-JNK (Thr183/Tyr185), p38 MAPK, and p-p38 MAPK (Thr180/Tyr182) were purchased from Cell Signaling Technology (Cell Signaling Technology, USA). Anti-β-tubulin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, USA).

Fresh purified NK cells were lysed in M-PER Mammalian Protein Extraction Reagent (Thermo). Then, samples were run on precast 4–12% Bis–Tris protein gels (Genscript). Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% w/v skimmed milk at room temperature for 1 h. Then, PVDF membranes were incubated with primary antibodies in 5% w/v skimmed milk in Tris-buffered saline containing 0.1% Tween-20 at 4 °C overnight, then incubated with Anti-rabbit IgG, HRP-linked Ab (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. To detect several proteins on the same PVDF membrane, membranes were processed with western stripping buffer (Beyotime) and reincubated with primary Abs. Chemiluminescence autoradiography was used to detect protein bands. The primary Abs for METTL3, WTAP, YTHDC2, YTHDC1, YTHDF1, ALKBH5, FTO, SHP2, AKT, p-AKT S473, STAT5, p-STAT5 Y694, Lamin B1, mTOR, p-mTOR Ser2448, p38 MAPK, p-p38 MAPK Thr180/Tyr182, and β-actin were purchased from Cell Signaling Technology; primary Ab SOCS3 was purchased from Abcam. The Anti-rabbit IgG, HRP-linker antibody was purchased from Cell Signaling Technology. The dilution of all antibodies used for immunoblotting was 1:1000.

Cells were lysed on ice for 30 min in lysis buffer (50 mM Tris-HCl (pH 8.0) 0.1% Triton X-100, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM PMSF, 1 mM sodium orthovanadate, and 2 mM leupeptin). After centrifugation, the protein concentration in the supernatant was determined by a BSA kit (Pierce, Rockford, IL, USA). Samples were separated by SDS-PAGE and transferred and were then immunoblotted with the following antibodies: p-p38 MAPK (Thr180/Tyr182), p38 MAPK, p-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, and p-Akt (Ser473) from Cell Signaling Technology (Beverly, MA, USA) and α-tubulin from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of whole cell lysates and western blotting analysis was performed as previously described.³⁴, ³⁵ Primary antibodies against cyclin A2 (1:1000), cyclin B1 (1:1000), cyclin D1 (1:1500), cyclin D2 (1:1500), cyclin D3 (1:1500), cyclin E1 (1:1000), CDK2 (1:1500), CDK4 (1:1500), CDK6 (1:1500), CDC2 (1:1500), β‐catenin (1:1500), GSK‐3β (1:2000), p‐GSK‐3β (Ser 9) (1:2000), p‐AKT (Ser 473) (1:2000), c‐jun (1:1500), c‐myc (1:1500), PCNA (1:1500), PARP (1:1500), cleaved‐PARP (1:1500), pro‐caspase 3 (1:1500), cleaved‐caspase 3 (1:1500), RIPK 1 (1:1500), p‐RIPK 1 (Ser166) (1:1500), ERK 1/2 (1:2000), p‐ERK (Thr202/Tyr204) (1:2000), p‐SAPK/JNK (Thr183/Tyr185) (1:1500) and p‐p38 MAPK (Thr180/Tyr182) (1:2000) were purchased from Cell Signalling Technology (Danvers, MA). An antibody against AKT (1:500) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and antibodies against β‐actin (1:2000) and β‐tubulin (1:2000) were acquired from Beijing Ray Antibody Biotech (Beijing, China). The grey values of proteins expression in Western blotting were quantified using the Image J software (NIH Image, Bethesda, MD).

Evodiamine was obtained from Matsuura Yakugyo Co Ltd (Nagoya, Japan). Primary antibodies against APP, p-GSK3β (Ser9), p-GSK3β (Tye216), p-SAPK/JNK (Thr183/Tyr185), p-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), and p-p38 MAPK (Thr180/Tyr182) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against p-tau (Ser396), JNK, ERK, and p38 were purchased from Abcam (Cambridge, MA, USA). Antibodies against p-tau (Ser202/Thr205), p-tau (Ser262), and tau were obtained from Thermo Fisher Scientific (Waltham, MA, USA); antibodies to GSK3β and GAPDH were obtained from GeneTex Inc. (Hsinchu city, Taiwan). Horseradish peroxidase (HRP)-conjugated antimouse and antirabbit IgG secondary antibodies were purchased from Jackson ImmunoResearch Inc. (West Grove, PA, USA). OA was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Unless otherwise stated, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Dulbecco’s Modified Eagle Medium (DMEM, 4.5 g/L D-glucose, L-glutamine and 110 mg/L sodium pyruvate), RPMI-1640 medium, fetal bovine serum, penicillin streptomycin (10,000 units/mL penicillin and 10,000 μg/mL streptomycin), and 0.25% trypsin-EDTA were obtained from Gibco, Thermo Fisher Scientific, Inc. (Waltham, MA, USA). GSPs (purity ≥ 95.0%) were obtained from Chengdu Must Bio-technology Co., Ltd. (Chengdu, Sichuan, China) and dissolved in dimethyl sulfoxide (DMSO). SB203580, PD98059 and SP600125 were obtained from Beyotime Biotechnology (Shanghai, China).
Antibodies against extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38 MAPK, NAG-1, cyclin-dependent kinase 1 (CDK1), cyclinB1, p21 and GAPDH were obtained from Proteintech Group, Inc. (Wuhan, Hubei, China). Antibodies against p-ERK (Thr202/Tyr204), p-JNK (Thr183/Tyr185) and p-p38 MAPK (Thr180/Tyr182) were obtained from Cell Signaling Technology (Boston, MA, USA).