Interaction of OnMBL with OnCD91a. (A) RNAi of OnMBL induced changes in expression of OnCD91a in vivo. After OnMBL RNAi for 12 h, the expression of OnCD91a was analyzed by qRT-PCR at 6 h and 12 h post–bacterial infection (n = 4). (B) After OnMBL RNAi for 12 h, the expression of OnCD91a was analyzed by Western blot at 12 h post–bacterial infection (mouse anti-tilapia CD91a pAb as the primary antibody, β-actin as the reference). (C) After OnMBL RNAi for 12 h, the OnCD91a level was detected by qRT-PCR at different time points postinfection with S. agalactiae in the presence or absence of OnMBL (n = 4). (D) After OnMBL RNAi for 12 h, the OnCD91a level was detected by Western blot at 12 h postinfection with S. agalactiae in the presence of Trx or OnMBL. (E) The mRNA expression of OnCD91a from tilapia head kidney MФ. The MФ were treated with S. agalactiae or A. hydrophila in the presence of OnMBL or PBS (n = 4). (F) Expression of OnCD91a in the indicated tissues was determined by qRT-PCR (n = 4). (G) Expression levels of OnCD91a in the liver, spleen, and head kidney were detected after bacteria challenge (n = 4). (H) Expression analysis of OnCD91a in MФ (n = 4). (I) Expression, purification, and Western blot analysis of OnCD91a. Line 1, analysis by SDS-PAGE (silver stain) of OnCD91a eukaryotic protein; line 2, Western blot analysis of OnCD91a eukaryotic protein was performed by preparing anti-His Ab; line 3, Western blot analysis of purified OnCD91a eukaryotic protein by preparing anti-CD91a pAb; lines 4 and 5, Western blot analysis of OnCD91a in tilapia head kidney and MФ lysate. (J) The purified OnCD91a eukaryotic protein was identified by MALDI-TOF. Peptides identified by mass spectrometry were shaded in gray. (K) The b/y ions of the representative peptide of purified protein were analyzed by LC-mass spectrometry/mass spectrometry. (L) Interaction analysis of OnMBL, OnCRT, and OnCD91a. The binding interface of protein ternary complex was analyzed by modeling with AlphaFold 3. Among them, based on CD91a as the interaction reference chain, the interaction details were supplemented by PyMOL v2.5.4. (M and N) Confirmation of OnMBL/CRT and OnCD91a interaction by ELISA. (O–Q) Interaction between the His-tag OnCD91a and OnMBL/CRT was detected by IP assay and shown by Western blot.