Fig. 5: NirP1 expression and pull-down analysis.

a Detection of NirP1 after 24 h of induction with 2 µM Cu₂SO₄ by Western blotting using anti-FLAG antiserum against tagged NirP1 in three biological replicates (R1–R3). The wild type was used as a control. b NirP1 pull-down analysis of two biological, independent replicates (R1 and R2). Upper panel: Coomassie-stained SDS gel, the NirP1 signal, and a band indicating a prominent coeluting protein of ~60 kDa are marked with arrows. Fractions are numbered and labeled CE cell extract, FT flow-through, W wash, E elution. Lower panel: Immunological identification of the lower band as NirP1. c Mass spectrometry-based analysis of NirP1 co-IP. Scatter plot of log₂-transformed LFQ protein ratios (NirP1-3xFlag co-IP/cell extract) from two independent replicates. Displayed are proteins with a higher abundance in NirP1-3xFlag co-IP elution fractions compared to the initial cell extract. The main interacting protein was identified as ferredoxin-nitrite reductase (NiR), which is encoded by the nirA/slr0898 gene. d Western blot of NirP1 co-IP fractions using native, denaturing, or reducing conditions. All samples were loaded in biological replicates R1 and R2. e NirP1-NiR (WP_010873675.1) interaction in Synechocystis 6803 predicted by AlphaFold^31,32 using their full-length sequences. The interaction modeled for the NiR and NirP1 homologs from Synechococcus 7942 is shown in Supplementary Fig. S7b. Source data are provided as a Source Data file.