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

Journal: Neural regeneration research

Article Title: Argatroban promotes recovery of spinal cord injury by inhibiting the PAR1/JAK2/STAT3 signaling pathway.

doi: 10.4103/1673-5374.375345

Figure Lengend Snippet: Figure 8 ｜Argatroban decreases GFAP expression and astrocyte activation for repairing SCI. (A) The whole-landscape immunofluorescence image of GFAP (green) at 6 weeks post-injury. DAPI was used to label cell nuclei (blue). Immunopositivity of GFAP in the argatroban group was reduced compared with the SCI group. A1, A2, and A3 represent the epicenter of spinal cord injury. A1’–A3’ represent the GFAP channel and A1’’– A3’’ represent the merged channel of GFAP and DAPI. DAPI was used to label nuclei (blue). Scale bars: 1 mm (A1–3), 25 µm (A1’–A3’ and A1’’–A3’’). (B) Quantification of fluorescence intensity of GFAP in (A). (C) Quantification of fluorescence intensity of GFAP in epicenter in A1, A2, and A3. Data are shown as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; SCI: spinal cord injury.

Article Snippet: Concentration Anti-thrombin Rabbit Abcam, Cambridge, MA, USA ab92621 AB_10711179 1:1000 (western blotting) Anti-PAR1 Rabbit Gene Tex, Irvine, CA, USA GTX64534 NA 1:1000 (western blotting) Anti-JAK2 Rabbit CST, Danvers, MA, USA 3230S NA 1:1000 (western blotting) Anti-STAT3 Mouse Abcam ab119352 AB_10901752 1:1000 (western blotting) Anti-pSTAT3 Rabbit Abcam ab76315 AB_1658549 1:1000 (western blotting) Anti-GAPDH Mouse Santa Cruz Biotechnology, Hercules, CA, USA sc-32233 AB_627679 1:500 (western blotting) Anti-Vimentin Rabbit Abcam ab92547 AB_10562134 1:200 (Immunofluorescence staining) Anti-GFAP Rabbit Bioss, Beijing, China bs-0199R NA 1:200 (Immunofluorescence staining) Anti-PAR1 Mouse Santa Cruz Biotechnology sc-13503 NA 1:200 (Immunofluorescence staining) Anti-rabbit IgG, HRP-linked antibody Goat CST 7074S NA 1:1000 (western blotting) Anti-mouse IgG, HRP-linked antibody Goat CST 7076S NA 1:1000 (western blotting) Fluorescent Alexa Fluor 488 goat anti-rabbit Goat Beyotime, Shanghai, China a0423 AB_2891323 1:200 (Immunofluorescence staining) Cy3-labeled goat anti-mouse IgG (H+L) Goat Beyotime a0521 NA 1:200 (Immunofluorescence staining) GADPH: Glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; HRP: horseradish peroxidase; JAK2: Janus kinase 2; NA: not applicable; PAR1: proteaseactivated receptor 1; pSTAT3: phosphor-signal transducer and activator of transcription 3; STAT3: signal transducer and activator of transcription 3.

Techniques: Expressing, Activation Assay, Immunofluorescence, Fluorescence

Journal: Neural regeneration research

Article Title: Argatroban promotes recovery of spinal cord injury by inhibiting the PAR1/JAK2/STAT3 signaling pathway.

doi: 10.4103/1673-5374.375345

Figure Lengend Snippet: Figure 7 ｜Argatroban inhibits astrogliosis at the acute phase of SCI. (A) Immunofluorescence image of Vimentin (green) and PAR1 (red) at 3 days post-injury. DAPI was used to label cell nuclei (blue). The expression of Vimentin and PAR1 in the argatroban group were reduced compared with the SCI group. Scale bar: 25 µm, 100 µm (enlarged images). (B, C) Quantification of fluorescence intensities of Vimentin and PAR1. (D) Diagram of the transverse spinal cord section. The box indicates the observation site in the spinal cord. Data are shown as mean ± SEM (n = 3). ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). DAPI: 4′,6-Diamidino-2- phenylindole; PAR1: protease-activated receptor 1; SCI: spinal cord injury.

Article Snippet: Concentration Anti-thrombin Rabbit Abcam, Cambridge, MA, USA ab92621 AB_10711179 1:1000 (western blotting) Anti-PAR1 Rabbit Gene Tex, Irvine, CA, USA GTX64534 NA 1:1000 (western blotting) Anti-JAK2 Rabbit CST, Danvers, MA, USA 3230S NA 1:1000 (western blotting) Anti-STAT3 Mouse Abcam ab119352 AB_10901752 1:1000 (western blotting) Anti-pSTAT3 Rabbit Abcam ab76315 AB_1658549 1:1000 (western blotting) Anti-GAPDH Mouse Santa Cruz Biotechnology, Hercules, CA, USA sc-32233 AB_627679 1:500 (western blotting) Anti-Vimentin Rabbit Abcam ab92547 AB_10562134 1:200 (Immunofluorescence staining) Anti-GFAP Rabbit Bioss, Beijing, China bs-0199R NA 1:200 (Immunofluorescence staining) Anti-PAR1 Mouse Santa Cruz Biotechnology sc-13503 NA 1:200 (Immunofluorescence staining) Anti-rabbit IgG, HRP-linked antibody Goat CST 7074S NA 1:1000 (western blotting) Anti-mouse IgG, HRP-linked antibody Goat CST 7076S NA 1:1000 (western blotting) Fluorescent Alexa Fluor 488 goat anti-rabbit Goat Beyotime, Shanghai, China a0423 AB_2891323 1:200 (Immunofluorescence staining) Cy3-labeled goat anti-mouse IgG (H+L) Goat Beyotime a0521 NA 1:200 (Immunofluorescence staining) GADPH: Glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; HRP: horseradish peroxidase; JAK2: Janus kinase 2; NA: not applicable; PAR1: proteaseactivated receptor 1; pSTAT3: phosphor-signal transducer and activator of transcription 3; STAT3: signal transducer and activator of transcription 3.

Techniques: Immunofluorescence, Expressing, Fluorescence