Rabbit anti p c jun

The patient cohort used herein consists of 47 EC, 20 SEH, 12 CEH and 38 specimens of normal endometrium that underwent surgery at the First Affiliated Hospital of Anhui Medical University (AMU) (Hefei, Anhui, People's Republic of China) between 2001 and 2002 as previously described [58 (link)]. The Institutional Review Board of AMU approved the protocol for the use of patient specimens in this study [58 (link)]. Patient consent forms were obtained from all patients in accordance with the Declaration of Helsinki [58 (link)]. IHC analysis was performed as previously described [58 (link)] using rabbit anti-TFF3 was obtained from Abcam, Cambridge, MA; rabbit anti-SP1, rabbit anti-p-c-JUN and rabbit anti-c-JUN antibodies wereobtained from Cell Signaling Technology, Singapore [17 (link)]. The details of the cohort and IHC scoring methodology have previously been described in detail [30 (link), 58 (link), 59 (link)].

The following reagents were obtained commercially. Rabbit anti-caspase-3, rabbit anti-P-JNK, and rabbit anti-P-c-Jun antibodies were from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-Flag, mouse anti-HA, mouse anti-β-actin antibodies, Nec-1, DPQ, PI, and 50% glutaraldehyde were from Sigma-Aldrich (Saint Louis, MO, USA). The mouse anti-FAF1 antibody has been described previously [31 (link)]. Mouse anti-COX IV, horseradish peroxidase (HRP)-conjugated anti-mouse, HRP-conjugated anti-rabbit, and fluorescein (FITC)-conjugated anti-mouse antibodies and 4′,6-diamidino-2-phenylindole (DAPI) were from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Mouse anti-JNK1 antibody and annexin V were from BD Biosciences (San Jose, CA, USA). Rabbit anti-GFP antibody was from Santa Cruz Biotechnology (Dallas, TX, USA). z-VAD-fmk was from Millipore (Darmstadt, Germany). SP600125 was from Tocris Bioscience (Minneapolis, MN, USA).

Brain tissues were fixed with 4% paraformaldehyde for 24 h and then incubated with 20% and 30% sucrose solutions for 24 h. Next, the brains were sectioned into 25 μm sections, and the sections were washed 3 times with a phosphate-buffered saline (PBS) solution. After washing, the sections were permeabilized with 1% Triton X-100 and blocked with donkey serum for 90 min, followed by incubation with primary antibodies overnight at 4 °C. The primary antibodies used included: rabbit anti-ASK1 (1:100, Abcam, US), goat anti-Iba1 (1:1000, Novus, US), mouse anti-NeuN (1:2000, Abcam, US), rat anti-CD16 (1:100, BD, US), rabbit anti-CD206 (1:200, Abcam, US), rabbit anti-pJNK (1:200, Affinity, China), rabbit anti-pp38 (1:200, Affinity, China), rabbit anti p–c jun(1:200, Cell Signaling Technology, US) and rat anti-CD68(1:500, Bio-Rad, US). The next day, the sections were incubated with secondary antibodies in the dark for 60 min at 37 °C. The following secondary antibodies were used: Alexa Fluor 594-labelled goat anti-rabbit IgG (Proteintech; 1:1000), fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Proteintech; 1:1000), and Alexa Fluor 647-labelled goat anti-mouse IgG (Proteintech; 1:1000). Finally, the immunofluorescently labelled sections were captured using a laser scanning confocal microscope (Nikon, Germany).

Co-Immunoprecipitation (co-IP) was performed with 3-day post-infection (95–100%) THP-1 cells using Pierce Protein A/G Agarose kit (Thermo Fisher Scientific) according to manufacturer’s protocol. Protein samples were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and blocked for 1 h at room temperature in Tris-buffered saline with 5% nonfat dry milk and 1% Triton-X 100. Primary antibodies included mouse anti-FBW7 (1:1000; R&D Systems, used to detect both unmodified and ubiquitinated FBW7), rabbit anti-TRP120 peptide antisera (1:10,000), rabbit anti-GAPDH (1:10,000; Proteintech, Rosemont, IL), rabbit anti-NICD (1:1000; Cell Signaling Technology, Danvers, MA), rabbit anti-p-c-Jun (1:1000; Cell Signaling Technology), rabbit anti-MCL1 (1:1000; Cell Signaling Technology), rabbit anti-K48 linkage-HRP conjugated (1:1000; Cell Signaling Technology), mouse anti-FK2 (1:500; Cell Signaling Technology), and mouse anti-cMYC (9E10) (1:500, Santa Cruz Biotechnology, Dallas, TX). Secondary antibodies included horseradish peroxidase-labeled goat anti-rabbit IgG and anti-mouse IgG (1:20,000; Kirkegaard & Perry, Gaithersburg, MD). Densitometry was performed using ImageJ software.