Ab222699

RIP experiment was conducted with Magna RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Generally, 80–90% confluent cells were collected and lysed with RIP lysis buffer, and the cell concentration was adjusted to 2.0 × 10⁷ cells/L. Then, 100 μL cell lysate was incubated by RIP buffer with magnetic beads conjugated with anti-FOSL2 (ab222699, Abcam) or negative control normal rabbit IgG (Cat. #PP64B, Millipore). The samples were incubated with proteinase K to digest the protein, then the immunoprecipitated RNA was isolated and analyzed by qRT-PCR. The primer sequence of FOSL2 was described as before.

The protein was extracted from the hippocampal tissues for western blot analysis. After determining the protein concentration using the BCA method, the proteins were processed in 12% sodium dodecylsulphate polyacrylamide gel electrophoresis. After transferring onto the PVDF membrane, the nonspecific staining was blocked by 5% defat milk. The membrane was incubated with the antibodies, including BDNF (1 : 1000, DF6387, Affinity), c-fos (1 : 1000, ab222699, Abcam), and β-actin (1 : 3000, ab8227, Abcam). Thereafter, the membranes were probed with anti-mouse immunoglobulin (Ig) G or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h.

The mice were anaesthetised with 2% isoflurane and transcardially perfused with phosphate-buffered saline (PBS, pH 7.4) and 4% paraformaldehyde until their limbs stiffened. The brains were fixed in 4% paraformaldehyde for at least 24 h at 4 °C and then immersed in 30% sucrose solution for another 24 h. A freezing microtome (CM1850, Leica, Hesse-Darmstadt, Germany) was used to prepare 20-µm-thick brain slices.
Then, brain slices were labelled with primary antibodies at 4 °C overnight, including C-Fos (1:500, mouse, GTX16902, Neobioscience, Shenzhen, China), NALCN (1:500, rabbit, ASC-022, Alomone, Jerusalem, Israel), and C-Fos (1:500, rabbit, ab222699, Abcam, Cambridge, UK). After overnight incubation, the primary antibody was washed with PBS and incubated with the secondary antibody, including Alexa Fluor 488 goat anti-mouse (1:500, ZF-0512, ZSGB-BIO, Beijing, China), Alexa Fluor 555 donkey anti-mouse (1:500, A-31570, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and Alexa Fluor 550 goat anti-rabbit (1:500, 550045, ZEN-BioScience, Chengdu, China) for 2 h. All images were taken with a Zeiss Axio Imager Z.2. and analysed using ImageJ software V1.8.0 (National Institutes of Health, Bethesda, MA, USA).

Immunofluorescence was used to detect the positivity rate of selected molecules in each group of cells. The cells were fixed with 4% paraformaldehyde for 15 min, After the cell antigen was repaired and penetrated by Triton X-100 for 10 min, the cells were incubated with the following primary antibodies against: IL1B (AF7209; Beyotime China), FOS (ab222699; Abcam, Waltham, MA, USA), JUN (ab40766; Abcam), FCGR2A (HPA010776; Atlas Antibodies, Sweden), SRC (ab47505; Abcam), and IL6 (SAB5700632; Sigma-Aldrich, Germany) at 37 °C for 2 h. Primary antibody dilutions were set as follows: IL-1B, 1:100; FOS, 1:100; JUN,1:100; FCGR2A, 1:200; SRC, 1:100; IL6,1:200. PC12 cells were incubated with FITC-labeled secondary antibody (Invitrogen, Waltham, MA, USA) and H9c2 cells were incubated with Alexa-labeled fluorescent secondary antibody (Invitrogen) at 37 °C for 1 h. Secondary antibody dilutions were set as 1:200. Nuclei were stained with 0.5 µg/ml concentration of DAPI (Southern Biotech, Birmingham, Alabama, USA). The cells were imaged with a fluorescent microscope (Nikon ECLIPSE Ni-U/DS-Ri2, Nikon Co., Ltd. Kanagawa, Japan). The positive cell rate (five randomly selected high-power fields of view with at least 100 cells/group) was calculated as number of positive cells/total number of cells × 100.

IHC was performed on paraffin-embedded sections of human gastric cancer tissues or tumor xenografts of nude mice using antibodies against c-JUN (1:50, ab40766, Abcam,), c-FOS (1:1000, ab222699, Abcam), Ki-67 (1:100, 27309-1-AP, Proteintech, Wuhan, China) and YAP1 (1:100, 13584-1-AP, Proteintech, Wuhan, China). The staining intensity and percentage of positive nucleuses was evaluated by two professional pathologists independently. The staining intensity was assessed as 0 (Negatively stained), 1 (weakly stained), 2 (moderately stained) and 3 (strongly stained). The IHC scores were calculated according to the formula: IHC score = P1 × 1 + P2 × 2 + P3 × 3 (P: percentage of positive nucleuses).

A Magna RNA-Binding Protein Immunoprecipitation kit (Millipore, Billerica, MA, USA) was applied to conduct the RIP assay. In brief, cells were lysed in RIPA lysis buffer, and the collected cell lysates were mixed with anti-AGO2 (ab222699; Abcam, Cambridge, UK) or IgG (Cat. #PP64B; Millipore) and magnetic beads. After digesting proteins by adding proteinase K, the quantitative Polymerase Chain Reaction (qPCR) was performed to assess the enrichment of BZW2.