Journal: bioRxiv

Article Title: Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death

doi: 10.1101/2024.03.01.582947

Figure Lengend Snippet: (A) N-104 killing assay of PA14 at 300 mOsm/l. (B) N-104 column capture of thrombin from pooled human basal tears, as per absence in the final wash fraction and detection in the KCl elution fractions. None bound to the negative control C-95 column. (C) GKY20 solution killing assay of PA14. Optimization of GKY20 concentration for checkerboard assay with N-104. (D) Replicates of N-104 checkerboard assays with GKY20 or each with peptides of gelsolin and cystatin S. (E) N-104, but not C-95, binding of immobilized thrombin peptide GKY20 as respectively detected with rabbit polyclonal ab-C-term or ab-N-term anti-lacritin antibodies. (F) N-104 and N-104 analog ligation of immobilized thrombin peptide GKY20 as detected with rabbit polyclonal ab-C-term anti-lacritin antibodies. (G) Ab-C-term antibody detects N-104 but not N-104 ‘L108S/L109S/F112S/L114S/L115S/W118S’ (’LFW:S’). (H) Since ab-C-term antibody cannot detect N-104 LFW:S, the capacity of increasing amounts of LFW:S to inhibit N-104 ligation of GKY20 was assessed. (I) N-104 versus LFW:S killing assay of PA14.

Article Snippet: Anti-thrombin western blotting of non-boiled flowthrough, final wash fraction and KCl elution fractions in 4x Laemmli buffer containing 10% β-mercaptoethanol was performed after separation on 4 - 20% gradient SDS-PAGE gels (4561094, Bio-Rad), transfer onto nitrocellulose (10600001, GE Healthcare Life Sciences), blocking with 1% fish gelatin (G7765, Sigma) in 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 2.7 mM KCl, 137 mM NaCl, pH 7.2 supplemented with 0.1% Tween-20 (1706531, Bio-Rad), and then incubation overnight (4°C) with 10 ml of 1 μg/ml of polyclonal rabbit anti-coagulation factor II/thrombin antibody (NBP1-58268, Novus Biologicals; antigen comprises amino acids 395 - 445) in 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 2.7 mM KCl, 137 mM NaCl, pH 7.2 supplemented with 0.1% Tween-20 (1706531, Bio-Rad) and 0.05% sodium azide.

Techniques: Negative Control, Concentration Assay, Binding Assay, Ligation