Phytochrome-interacting factors (PIFs) are a basic helix-loop-helix family of transcriptional regulators that maintain skotomorphogenesis and suppress photomorphogenesis. PIFs are regulated by plant photoreceptors, especially phytochromes. In general, PIFs physically interact with phytochromes, and this interaction induces PIF’s phosphorylation and subsequent degradation, contributing to the initiation of photomorphogenic development. Among the eight members of PIF (PIF1 to PIF8) reported in Arabidopsis thaliana, PIF3 is the first discovered member and plays central roles in de-etiolation and chlorophyll biosynthesis. More recently, PIF3 has been also reported to regulate hormone signaling and cold tolerance in plants. Although PIF3 protein shows dynamic behaviors in plants, its study has been limited due to the lack of an authentic PIF3 antibody. In this study, we produced polyclonal antibodies using inclusion bodies and characterized the PIF3 antibody based on specificity and sensitivity. In addition, we investigated PIF3 phosphorylation and degradation during phytochrome-mediated light signaling in plants. Furthermore, we successfully performed in vitro protein–protein interaction and co-immunoprecipitation assays between phytochrome B (phyB) and PIF3 using the antibody. Therefore, we obtained an authentic PIF3 antibody that could be used as a valuable tool to study the multi-faceted functions of PIF3.

植物色素相互作用因子（PIF）是转录调节因子的基本螺旋-环-螺旋家族，可维持拟态形成和抑制光态形成。PIF受植物光感受器，尤其是植物色素的调节。通常，PIF与植物色素发生物理相互作用，这种相互作用诱导PIF的磷酸化和随后的降解，从而促进了光形态发生的发展。在拟南芥中报道的八个PIF成员中（PIF1至PIF8），PIF3是第一个被发现的成员，并且在去黄酮化和叶绿素生物合成中起着核心作用。最近，还报道了PIF3调节植物中的激素信号传导和耐寒性。尽管PIF3蛋白在植物中表现出动态行为，但由于缺乏可靠的PIF3抗体，其研究受到了限制。在这项研究中，我们使用包涵体生产了多克隆抗体，并根据特异性和敏感性对PIF3抗体进行了表征。此外，我们调查了植物色素介导的光信号传导过程中PIF3的磷酸化和降解。此外，我们使用该抗体成功进行了植物色素B（phyB）和PIF3之间的蛋白质间相互作用和免疫共沉淀试验。因此，