Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Natterin bridges IFN-φ1 and non-canonical inflammasome pathways via CRFB1 /Gbp4 to license Caspy2-mediated antibacterial immunity

doi: 10.3389/fcimb.2025.1686758

Figure Lengend Snippet: Effectiveness of CRISPR/Cas9 depletion of loc795232. Basal expression of loc795232 mRNA transcripts in unstimulated WT or KO embryos ( n = 100/group) at 1 dpf by RT-qPCR (A) or by in situ hybridization (B) . All qPCR data normalized to β-actin and expressed as fold change relative to 0h WT unstimulated control. Data represent mean ± SEM of three biological replicates. Independent groups of 1 dpf embryos previously treated or not with Pam3CSK4 or MCC950 and subjected to ST stimulation for 2h, as well as KO larvae stimulated with ST were processed for Natterin detection by WB (C) using anti-Natterin serum (62 kDa dimeric form) and anti-IgG TrueBlot HRP. The corresponding bar graph represents the protein expression levels as a percentage of the control (D) , and Ponceau S staining is visualized in the first horizontal line.

Article Snippet: The specific proteins were detected in the iBindTM Flex Western System using iBind Flex Cards (Invitrogen, #SLF2010) and the solution made with the iBind solution Kit (Invitrogen, #SLF1020) and the primary antibodies: rabbit polyclonal IgG anti-mouse caspase-1 (p-10, M-20) (#sc-514, Santa Cruz Biotechnology, Dallas, United States, 1/200, detects the immature form with 50 kDa and the mature form around 28 kDa), rabbit polyclonal IgG raised against amino acids 301–350 of caspase-11 of mouse origin (p-10 (M-50; #sc-28231; Santa Cruz Biotechnology, 1/200, detects the immature form with 50 kDa and the mature form around 28 kDa) followed by rabbit HRP-labeled anti-rabbit IgG TrueBlot second antibody (Rockland, #18-8816–33 at 1/1,000) for 3h.

Techniques: CRISPR, Expressing, Quantitative RT-PCR, In Situ Hybridization, Control, Staining