Normal mouse igg antibody

ChIP was performed using the Simple ChIP Enzymatic Chromatin IP Kit (#9003, Cell Signaling Technology) according to the manufacturer's protocol.^[23, ^28] In brief, single‐cell suspensions of spinal dorsal horn tissues were prepared using a glass tissue homogenizer. Then, cells were crosslinked with formaldehyde (final concentration 1%) for 10 min, and the cross‐linking was quenched by the addition of glycine (final concentration 0.125 m). Cells were then lysed, chromatin was harvested and fragmented using micrococcal nuclease digestion, and nuclear membranes were broken by sonication. The disposed chromatin was subjected to immunoprecipitation with normal mouse IgG antibody (Cell Signaling Technology, catalog #5415) as the negative control, rabbit histone H3 antibody (Cell Signaling Technology, catalog #14269) as the positive control, and NFAT1 mouse monoclonal antibody (Abcam, catalog #ab2722). The ChIP‐enriched DNA samples were quantified by quantitative ChIP‐PCR using specific primer pairs (Table S5, Supporting Information), and the PCR products were visualized by agarose gel electrophoresis.

Lung tissues and cells were lysed using IP lysis buffer (Sangon). Protein concentrations were determined using the bicinchoninic acid assay. An appropriate amount of primary antibodies (anti-O-GlcNAc antibody, PTM BioLabs [4 µg], Parkin antibody from Santa Cruz [4 µg], and normal mouse IgG antibody from Cell Signaling Technology [4 µg]) were added to 1000 µg of total protein and the samples were slowly shaken on a rotary shaker at 4 °C overnight. Then, 35 µL of protein A/G agarose beads (Invitrogen) was added to the samples, which were again slowly shaken on a rotary shaker at 4 °C for 2 h. Next, the beads were washed thrice with the lysis buffer, the liquid was discarded, and ddH₂O and 4 × protein loading buffer were added to the samples, which were then heated at 100 °C for 10 min. Finally, after centrifuged at 4 °C and 1000 rpm for 1 min, the supernatants were collected and subjected to western blot analysis.