Mettl14

An RIP Kit (IEMed-K303, IEMed) was used for the RNA immunoprecipitation (RIP) assay. Tissues of DRGs (0.1–0.2 g) were collected for each sample and then mechanically sheared in RIP lysis buffer containing a protease inhibitor cocktail and RNase inhibitor using a homogenizer. After centrifugation (4°C, 15 minutes, 12,000g), the supernatant was collected and split into 3 fractions: input, IP, and IgG. Antibodies against m6A (3 μg; Synaptic Systems), METTL14 (3 μg; ABclonal), normal rabbit IgG, and Protein A/G beads were added to the supernatant and incubated at room temperature for 1 hour. Beads were collected and washed 5 times with RIP washing buffer, and the RNA was eluted from the beads by addition of 20 μL RNase-free water. Purified RNA was subjected to qPCR to determine the presence of binding targets.

ATF4 (rabbit, 11815S), p-mTOR (rabbit, 5536S), mTOR (rabbit, 2983S), p-p70S6K (rabbit, 9205S), p70S6K (rabbit, 2708S), pS6 (rabbit, 4857S), LC3B (rabbit, 3868S), and sequestosome 1 (SQSTM1, rabbit, 8025) monoclonal antibodies were purchased from Cell Signaling Technology (Shanghai, China). DDIT4 (rabbit, 10638-1-AP) and YTHDF2 (rabbit, 24744-1-AP) polyclonal antibodies were purchased from Proteintech. FTO (rabbit, ab126605), WTAP (rabbit, ab195380), ALKBH5 (rabbit, ab69325), and m⁶A (mouse, ab208577) antibodies were purchased from Abcam. METTL3 (rabbit, a8370), and METTL14 (rabbit, a8530) polyclonal antibodies were purchased from ABclonal. Compound 968 was purchased from Sigma-Aldrich (SML1327). Actinomycin D was purchased from Selleck Chemicals (S8964). Chloroquine (CQ) was obtained from Sigma-Aldrich (C6628). Meclofenamate sodium was purchased from MCE (HY-B1320). CB839 was purchased from Selleck Chemicals (S7655).