Anti phospho sapk jnk thr183 tyr185

All cell culture materials were obtained from Gibco Inc. Recombinant Human Activin-A (GFH6) was purchased from Cell Guidance Systems. RepSox (#72392), a cell permeable, selective inhibitor of the TGF-β type 1 receptor (TGFβRI) ALK5 was obtained from Stemcell Technologies. Recombinant forms of FSH (Gonal-F) and hCG (Ovitrelle) was purchased from Merck Global (Darmstadt, Germany). SAPK/JNK Kinase Assay Kit (#8794, nonradioactive), Hoechst 33342 (#4082), anti c-Jun (#9165), anti-phospho-c-Jun^Ser73 (#3270S), Anti-Phospho-SAPK/JNK^{Thr183/Tyr185} (#9251), Smad2 (#3122) and Phospho-Smad2^{Ser465/Ser467} (#18338) antibodies were obtained from Cell Signaling. Oil Red O was purchased from Sigma Inc. (USA). All western blotting buffers and reagents were purchased from Bio-Rad. Anti-Vinculin Antibody (V9264) was purchased from Sigma-Aldrich. Mouse antihuman monoclonal antibodies were purchased from Santa Cruz Biotechnology for the detection of human 3β-HSD Type II (sc-100466), 17β-HSD type-I (sc-376719), StAR (sc-166821), and P450 SCC (CYP11A1, sc-292456). Aromatase (CYP19A1, ab34193) monoclonal mouse antibody was from Abcam Inc. YO-PRO®-1 Iodide (491/509) was obtained from Life Technologies. Texas Red™-X Phalloidin was obtained from Thermo Fisher Scientific.

Human CXCL5/ENA-78 quantikine enzyme-linked immunosorbent assay (ELISA) kit (DX000), recombinant human CXCL5/ENA-78, and anti-CXCR2/CXCL8RB and anti-CXCL5 were purchased from R&D (Minneapolis, MN, USA). SYBR Premix Ex Taq was from TaKaRa (Shiga, Japan). Matrigel™ Basement Membrane Matrix was from BD Bioscience (Franklin Lakes, NJ, and USA). Lipofectamine™ 2000 Transfection Reagent was from Invitrogen (Grand Island, NY, USA). Anti-p44/42 MAPK(Erk1/2), anti–phospho-p44/42 MAPK(Erk1/2) (Thr202/Tyr204), anti-p38 MAPK, anti–phospho-p38 MAPK (Thr180/Tyr182), anti-SAPK/JNK, anti–phospho-SAPK/JNK (Thr183/Tyr185), and ERK1/2 inhibitor U0126 were from Cell Signaling Technology (Boston, MA, USA). CXCR2 inhibitor SB225002 was obtained from Calbiochem (Darmstadt, Germany).

The cells were washed twice with ice-cold PBS and scraped into RIPA buffer plus protein inhibitors. The protein concentration was determined as previously reported by Bradford MM et al., 1976 [33 ]. Proteins were separated on 10 or 12% SDS-PAGE and transferred to polyvinylidene difluoride filters. Membranes were blocked and probed with the following antibodies: anti-phospho-AKT (Thr308) (Cell Signaling), anti-total AKT (Santa Cruz Biotechnology, INC), anti-Tubulin (Sigma), anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-total SAPK/JNK (56G8) (Cell Signaling), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10), anti-p44/42 MAPK (Erk1/2) (137F5) (Cell Signaling), anti phospho-Ser307-IRS1, anti-total IRS1 (Millipore), anti-p65-NF-κB (C-20) (Sigma-Aldrich®), anti-Lamin B Receptor (abcam®) and anti-sXBP1 (R&D biosystems).
The protein band density was measured using the ImageJ 1.45 software (National Institutes of Health, Bethesda, MD). The amount of protein under control conditions was assigned a relative value of 100%.

Cell lysis, protein extraction, and western blot analyses were performed as described in our previous work (40 (link)). Proteins were dissolved in a lysis buffer and separated using SDS/PAGE for western blot analyses. Primary antibodies included rabbit anti-Phospho-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, anti-Phospho-c-Jun (Ser73), anti-c-Jun, anti-ATG5, anti-LC3B and anti-GAPDH (Cell Signaling Technology). Secondary antibody was HRP-conjugated anti-rabbit IgGs (Life Technologies). The densitometric analyses of western blotting images were performed using ImageJ software (National Institutes of Health).

Standard recombinant DNA techniques were used to construct pCS2/FLAG-rat Fz2 (FLAG-Fz2) and pPGK-neo/Wnt5a. pSuper-retro-GFP-Neo-shWnt5a, which was used for the generation of cells stably expressing Wnt5a shRNA, was generated by inserting small hairpin RNA against Wnt5a (5′-GTGGATAACACCTCTGTT-3′) into the pSuper-retro-GFP-Neo vector (Oligo Engine, Seattle, WA). The small interfering RNAs (siRNAs) used in this study are listed in Table S1. Wnt5a was purified to near homogeneity using three chromatography steps as described previously16 (link)55 (link). Anti-Wnt3a, anti-Wnt5a/b, anti-Src, anti-Fyn, anti-Yes, anti-phospho-Src family (Tyr416), anti-AKT, anti-phospho-AKT (Ser473), anti-phospho-PKC (pan) (Ser660), anti-SAPK/JNK, and anti-phospho-SAPK/JNK (Thr183/Tyr185) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-Rac1, anti-EEA1, anti-HSP90, anti-PKCα, and anti-Clathrin antibodies were from BD Biosciences (San Jose, CA). Anti-FLAG M2 and anti-Ror2 antibodies were from Sigma-Aldrich (Steinheim, Germany) and R&D Systems (Minneapolis, MN), respectively.

Western blot analysis was performed as described previously [21 (link)]. The primary antibodies used for Western blot analyses included anti-phospho-Src (Tyr418) (Invitrogen, Carlsbad, CA, USA), anti-phospho-FAK (Tyr576) (Invitrogen), anti-FAK (Invitrogen), anti-GAPDH (Invitrogen), anti-phospho-EGFR (Tyr1068) (Cell Signaling, Beverly, MA, USA), anti-phospho-STAT3 (Tyr705) (Cell Signaling), anti-phospho-PI3K (Tyr458) (Cell Signaling), anti-AKT (Cell Signaling), anti-phospho-SAPK/JNK (Thr183/Tyr185) (Cell Signaling), anti-SAPK/JNK (Cell Signaling), anti-phospho-Paxillin (Tyr118) (Cell Signaling), anti phosphor-p130Cas (Tyr410) (Cell Signaling), anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT3 (Santa Cruz Biotechnology), anti-PI3K (Santa Cruz Biotechnology), anti-phospho-MEK1/2 (Ser218/Ser222) (Santa Cruz Biotechnology), anti-MEK (Santa Cruz Biotechnology), anti-phospho-ERK (Tyr204) (Santa Cruz Biotechnology), anti-ERK2 (Santa Cruz Biotechnology), anti-Paxillin (Santa Cruz Biotechnology), anti-p130 Cas (Santa Cruz Biotechnology), anti-phospho-AKT (Ser473) (Millipore, Billerica, MA, USA)

Cellular protein was isolated by lysis in RIPA buffer (Boster, Wuhan). Proteins in all samples were quantified with Bicinchoninic acid protein assay. Proteins of equal amounts from all samples were separated with SDS/PAGE gel (Bio-Rad, United States) and transferred onto PVDF membrane (Bio-Rad, United States). Bands were sealed with 5% skim milk, incubated in primary antibodies at 4°C overnight, then in secondary antibodies at room temperature for 1 h, and then examined and analyzed by using ChemiDoc™ XRS+ with Image Lab™ Software (Bio-Rad, United States). Following antibodies were purchased: anti-caspase-3 (#9662), anti-PARP (#9532), anti-Phospho-SAPK/JNK (Thr183/Tyr185) (#4688), anti-Phospho-p38 MAPK (Thr180/Tyr182) (#4511), anti-HDAC1 (#34589), anti- HDAC2 (#57156), and anti-Histone H3 (#4499) were purchased from Cell Signaling Technology (Beverly, MA, United States); anti-GAPDH (60004-1-Ig) was purchased from Proteintech Group (Chicago, IL, United States); anti-Phospho-ERK (AP0472), anti-Phospho-MEK (AP1021) and anti-CDK6 (A0705) were purchased from ABclonal Technology (Wuhan, China); anti-cyclin D1 (YT1172) was purchased from Immunoway Biotechnology Company (TX, United States); goat anti-rabbit and goat anti-mouse were obtained from Boster Biological Technology (Wuhan, China).