P mapk

Proteins were extracted from the ovary tissues of mice using RIPA buffer containing protease inhibitors. Total protein was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were then incubated with the following specific primary antibodies obtained from Cell Signaling Technology (Danvers, MA, USA): TP53 (rabbit, 1:1000), p-AKT (rabbit, 1:1000), AKT (rabbit, 1:1000), p-MAPK (rabbit, 1:1000), and MAPK (rabbit, 1:1000). The membranes were then incubated with HRP-conjugated secondary antibodies. Eventually, the protein bands were photographed and images were developed.

Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 0.2 mM PMSF). Proteins (20–40 µg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, IPVH00010). After the blocking procedure, membranes were incubated with primary antibodies (1:1000) and HRP-conjugated secondary antibodies (1:5000), and visualized in Imager (Bio-Rad) using ECL system (Thermo Fisher Scientific, 34095). Antibodies used were: PD-L1 (R&D, MAB1086), NKG2D (R&D, MAB139), PD-1 (R&D, MAB1086), p-JAK1 (Y1022, Assay Biotech, A7125), p-JAK2 (Y1007 + 1008, Abbomax, 601–670), JAK1 (Abgent, AP20699a), JAK2 (Abgent, AP20700c), p-Stat1 (S727, Millipore, 07–714), Stat1 (Abgent, AP19835Bb), p-Stat3 (Y705, Abcam, ab76315), Stat3 (Abcam, ab5073), p-Stat5 (Y694, Abcam, ab32364), Stat5 (Abcam, ab16276), p-MAPK (Cell Signaling, 9101 S), p-Erk (Cell Signaling, 4695), p-Akt (S473, Cell Signaling, 9271), p-NFκB (S536, Abcam ab86299), and GAPDH (Cell Signaling, 2118 S).

Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 0.2 mM PMSF). Proteins (20-40 μg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking procedure, membranes were incubated with primary antibodies (1:1000), HRP-conjugated secondary antibodies (1:5000), and visualized in Imager (Bio-Rad) using ECL system (Thermo Fisher Scientific, 34095). Antibodies used were; PD-L1 (R&D, MAB1086), p-JAK1 (pY1022, Assay Biotech, A7125), p-JAK2 (pY1007 + 1008, Abbomax, 601-670), p-MAPK (cell Signaling, 9101S), p-Erk (Cell Signaling, 4695), p-MEK (Ser 217/221, Cell Signaling, 9121), p-Akt (S473, Cell Signaling, 9271), and p-Stat3 (pY705, Abcam, ab76315), p-Stat1 (pY701, Abbomax, 620-160), p-Stat5 (Abcam, pY694, ab32364), p-NFκB (S536, Abcam ab86299), p-mTOR (Millipore, 09-345), JAK1 (Abgent, AP20699a), JAK2 (Abgent, AP20700c), Stat1 (Abgent, AP19835b), MEK (Ameritech, ATB-T2715), MAPK (Signalway, 21245), ERK (Ameritech, ATB-T5371), Akt (Santa Cruz, sc5298) and GAPDH (Cell Signaling, 2118S).

Cells were lysed in a sample buffer containing 2% SDS, 60 mmol/L Tris‐HCl (pH 6.8) and 5% glycerol. Cell lysates were boiled for 5 minutes. Protein concentration was determined using a BCA kit (Beyotime, Shanghai, China), and equal amount of protein was loaded for Western blot analysis as previously described.¹¹ Primary antibodies against ERK1/2, p‐ERK1/2, p38, p‐p38, JNK1/2, p‐JNK1/2, PKCδ, p‐PKCδ, PKA, p‐PKA, MARK2, p‐MAPK, LKB1, p65 and p‐p65, as well as secondary antibodies, were all from Cell Signaling Technology (Boston, MA, USA). Anti‐β‐actin antibody was from Proteintech (Wuhan, China). Blots were developed using enhanced ECL chemiluminescence reagents (Pierce, Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instruction. β‐actin was used as a loading control.