Anti c jun antibody

The chromatin immunoprecipitation assay was performed according to the manufacturer’s protocol (Upstate Biotechnology, Lake Placid, NY, USA). Briefly, AML12 cells were treated with 1 μg of LPS for 24 h, fixed with 1% formaldehyde and then collected. Soluble chromatin was immunoprecipitated with an anti-c-Jun antibody (#9165, Cell Signaling Technology). After recovering DNA, PCR was performed using primers encompassing the AP-1-binding region on the mouse hepcidin promoter (forward 5′-CTGGCTGTAGGTGACACAAC-3′ and reverse 5′-AAGGACTTGTGTGGTGGCTG-3′). The size of the amplified PCR product was 193 bp.

ChIP analysis was performed using a ChIP assay kit (catalog: 17-371; Millipore, Billerica, MA, USA) according to the manufacturer’s instructions to determine whether c-JUN binds to the promoter of miR-4652-3p. First, cells were transfected with a c-JUN plasmid and fixed with 1% formaldehyde. The cross-linked DNA is sonicated, cut to a length of 200–1000 base pairs, and then subjected to an immunoselection process, which requires the use of an anti-c-JUN antibody (1:50; Cell Signaling Technology (CST), Inc., Danvers, MA, USA). Ultimately, the agarose gel electrophoresis results reveal whether the DNA fragment of the putative c-JUN binding site was present in the miR-4652-3p promoter.

ChIP assays were performed according to the instruction of Magna ChIP™ G Tissue Kit (Merk‐Millipore). Chromatin extracted from mouse colonic smooth muscle tissues was immunoprecipitated for 24 hrs at 4°C using anti‐c‐Jun antibody (Cell Signaling Technology, Danvers, MA, USA). One‐hundredth of the solution collected before adding the antibody was used as an internal control for the amount of input DNA. As a negative control, the antibody or DNA was omitted or replaced with normal rabbit IgG (Cell Signaling Technology). PCR was carried out using 2 × PCR Reagent (TIANGEN, Beijing, China), consisting of 3 min. at 94°C, 32 cycles of 20 sec. at 94°C, 30 sec. at 59°C and 30 sec. at 72°C and 2 min. at 72°C. Primers are shown in Table S3.

The following primary antibodies were used in this study: anti-USP14 antibody (#11931, 1:1000), anti-JNK antibody (#9252, 1:1000), anti-C-Myc antibody (#18583, 1:1000), anti-Cyclin D2 antibody (#3741, 1:1000), anti-MDM2 antibody (#86934, 1:1000), and anti-C-Jun antibody (#9165, 1:1000), which were obtained from Cell Signaling Technology (Beverly, MA, USA) and anti-Flag antibody (M185-3L), anti-HA antibody (561), anti-C-Myc antibody (M192-3), anti-β-actin antibody (M177-3) and anti-GAPDH (M171-3), which were obtained from MBL (Nagoya, Japan).

The cells (2 × 10⁷) were fixed with 1% formaldehyde and then stopped by 0.125 M glycine. The protein-bound chromatin was sheared into small fragments by sonication, and the specific protein/DNA complexes were immunoprecipitated at 4 °C overnight with an anti-c-Jun antibody (#9165S, Cell Signaling Technology, Danvers, MA, USA; 1:100) for ChIP or normal rabbit IgG (#2729, Cell Signaling Technology, USA, 1:100) as a control. Protein A/G PLUS-agarose (#sc-2003, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:50) was then supplemented and incubated at 4 °C for 6 h. After immunoprecipitation, DNA was decrosslinked, the proteins were removed by incubation with proteinase K, and the DNA was purified for qRT-PCR using primers listed in Supplementary Table 5.

MDA-MB-231, MDA-MB-468, and MC3T3-E1 cells treated were cultured as described above with or without OC for 2 weeks. The cells were then lysed with CHAPS buffer (Cell Signaling Technology, MA, USA). As a positive control for PLAP and TNAP, HeLa cells were cultured for 3 days and their protein extracted. The cell lysates were heated at 100 °C for 5 min with SDS sample buffer, and 10 µg of protein was electrophoresed in 10% acrylamide gel at 150 V for 1 h. The gel was blotted to a cellulose nitrate membrane at 150 mA. Anti-PLAP antibody (EPR6141, Abcam, Cambridge, UK) diluted 1,000 times, anti-TNAP antibody (EPR4477, Abcam) diluted 4,000 times, anti-c-Jun antibody (9165S, Cell Signaling Technology; diluted 2,000 times), anti-phospho-c-Jun antibody (3270S, Cell Signaling Technology; diluted 2,000 times), and anti-β-actin antibody (8H10D10, Cell Signaling Technology) were used as the primary antibodies. Peroxidase AffiniPure goat anti-rabbit IgG (H + L) (111-035-144, Jackson ImmunoResearch, West Grove, PA, USA) diluted 2,000 times for PLAP, TNAP, c-Jun, and phospho-c-Jun and anti-mouse IgG horseradish peroxidase (GE Healthcare, IL, USA) diluted 1,000 times for β-actin were used as secondary antibodies. After adding Thermo Pierce Western Blotting Substrate (Thermo Fisher Scientific), images were acquired with chemiluminescence detection using ChemiDoc Touch (Bio-Rad, CA, USA).