Antibody against flag

Then, 2 × 10⁷ CD34⁺ cells sorted from human cord blood containing the plasmids LEGO-iG2, LEGO-iG2-Flag-MH-fusion, or LEGO-iG2-Flag-MEF2D were collected. ChIP DNA was prepared by using the ChIP-IT High Sensitivity Kit (Active Motif). Immunoprecipitation was performed with the antibody against Flag (Proteintech). The ChIP-seq DNA libraries were constructed by using the VAHTS Universal Pro DNA Library Prep Kit (Vazyme) according to the manufacturer’s instructions. The libraries were sequenced on the NovaSeq 6000 (Illumina). For data analysis, we used the nextflow-based workflow nf-core/chipseq (version 1.2.1) to analyze raw ChIP-seq reads with the command “nextflow run nf-core/chipseq –input sampleinfo.csv -profile singularity –fasta GRCh38.primary_assembly. genome.fa–150 single_end –gtf gencode.v38.annotation.gtf –macs_gsize 2.7e9 –skip_fastqc –save_macs_pileup.”²⁵ (link) The core pipeline steps include: (1) adapter trimming, alignments of the raw reads using bwa; (2) mark duplicated reads using picard; (3) filtration and removing background reads using samtools, bedtools, and pysam; (4) creating normalized bigWig alignments using bedtools and bedGraphToBigWig for visualizations; (5) calling of broad/narrow peaks using MACS2; and (6) motif annotation of binding peaks using HOMER. More detailed information of the pipeline is available at https://github.com/nf-core/chipseq.

Wild-type (WT) NLRP12 complementary DNA (cDNA) sequences were obtained from NLRP12 clone plasmid by polymerase chain reaction (PCR), fused with a FLAG tag and inserted into pLVX-IRES-ZsGreen1 vector. Mutant NLRP12 constructs were generated by site-directed mutagenesis to introduce the p.F303Sfs∗25 and p.G729R substitutions according to the manufacturer's instruction. Human embryonic kidney 293T (HEK293T) (2 × 10⁵) in a 12-well plate were transfected by using Lipofectamine™ 3000 (Invitrogen, USA) with 1 μg expression plasmid. The expression of NLRP12 was examined by Western blot with an antibody against FLAG (Proteintech, USA).

HEK293 cells (1 × 10⁵) in 24-well plate were transfected with 200 ng of pNF-κB-LUC luciferase reporter (Byotime, China), 50 ng of Renilla luciferase reporter (Byotime, China), and 250 ng of NLRP12 expression plasmids or empty vector as indicated. The NF-κB signaling pathway was induced by transfection of 200 ng of p65. After 24 h of transfection, cells were treated with 20 ng/mL TNF-α or not for 16 h. Luciferase activities were determined on cell lysates (Dual-Luciferase® Reporter Assay System, Promega, USA). Using the same cell lysates, the NLRP12 protein tagged with FLAG were quantitated by Western blot with an antibody against FLAG (Proteintech, USA).