Journal: PLOS ONE

Article Title: IL-12p40 is essential but not sufficient for Francisella tularensis LVS clearance in chronically infected mice

doi: 10.1371/journal.pone.0283161

Figure Lengend Snippet: Splenocytes from 5 naïve mice or 5 mice vaccinated with 10 5 LVS i.d. were co-cultured with LVS-infected BMDMs from the same genotype. Bacterial CFU were assessed immediately after infection (Day 0) and three days after infection (A). Decreasing numbers of splenocytes were added to a constant number of infected BMDMs, where 1 = 5 x 10 6 splenocytes. Intramacrophage bacterial growth was assessed three days after infection (B). Supernatants pooled from three wells per group were collected on day 3 after infection from the experiment illustrated in (A) for analyses of IFN-γ (C), IL-12p40 (D), GM-CSF (E), nitric oxide (F), and TNF-α (G). Data shown are averages of three independent experiments (A–F) or one experiment (G). Mice used were ages 3–9 months and were aged matched within each experiment. *p < 0.05; ** p < 0.01; *** p < 0.001 by ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Membranes were blocked with 5% BSA in TBST for 1 hour at room temperature, then incubated in a 1/1500 dilution of rat anti-mouse IL-12p40 Ab (C15.6, BD PharMingen, San Diego, CA) in TBST overnight at 4°C.

Techniques: Cell Culture, Infection

Journal: PLOS ONE

Article Title: IL-12p40 is essential but not sufficient for Francisella tularensis LVS clearance in chronically infected mice

doi: 10.1371/journal.pone.0283161

Figure Lengend Snippet: The indicated mice were given 2 x 10 8 pHAGE-Il12b2 lentivirus or PBS i.p. on day 0, then challenged with 5 x 10 2 LVS or PBS i.d. on day 2 (A). Mice were then given two additional treatments of 10 7 lentivirus or PBS i.p. at days 3 and 7. Bacterial burdens were determined on day 11 (B) and IL-12p40 expression was determined by ELISA in spleen, liver, lungs (C), and sera (D). Data are from a representative of five independent experiments of similar design. Mice used were ages 4–9 months and were aged matched within each experiment. * p < 0.05; ** p < 0.01; *** p < 0.001 between KO + p40 and WT by ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Membranes were blocked with 5% BSA in TBST for 1 hour at room temperature, then incubated in a 1/1500 dilution of rat anti-mouse IL-12p40 Ab (C15.6, BD PharMingen, San Diego, CA) in TBST overnight at 4°C.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Free radical biology & medicine

Article Title: New insights in the molecular regulation of the NADPH oxidase 2 activity: Negative modulation by Poldip2.

doi: 10.1016/j.freeradbiomed.2023.02.019

Figure Lengend Snippet: Fig. 3. Comparison of gp91phox, Poldip2 and p22phox expression in monocytes, neutrophils and macrophages isolated from healthy donor blood. M0: monocyte; N: neutrophil; MA1 and MA2 macrophage type 1 (pro- inflammatory and anti-tumoral) and 2 (pro-tumoral and anti-inflammatory). 12 μg of total protein (cell lysates) are loaded. The ponceau staining of these western blots is shown in Fig. S5. All samples are from the same donor. Experiment was repeated independently twice with similar results for blood from two healthy donors.

Article Snippet: The membranes were incubated overnight at 4 ◦C with specific monoclonal or polyclonal antibodies: anti-gp91 (anti-gp91phox; 54.1; mouse monoclonal, dilution 1:1500; Santa Cruz), anti-p22 (44.1; mouse monoclonal; dilution 1:1500; Santa Cruz); anti-Poldip2 (ab181841; rabbit monoclonal; , dilution 1:1500; Abcam), anti-p67 (07-002; rabbit polyclonal; dilution 1:1000; Millipore); Anti-p47 (BD 610354; rabbit polyclonal; dilution 1:1500; Bioscience), anti-Rac1 (ARCO3; mouse monoclonal; dilution 1:500; Cytoskeleton), anti-NOX4 (ab109225; rabbit monoclonal; dilution 1:1500; Abcam).

Techniques: Comparison, Expressing, Isolation, Staining, Western Blot

Journal: Free radical biology & medicine

Article Title: New insights in the molecular regulation of the NADPH oxidase 2 activity: Negative modulation by Poldip2.

doi: 10.1016/j.freeradbiomed.2023.02.019

Figure Lengend Snippet: Fig. 5. Poldip2 interaction with purified membrane fraction isolated from human neutrophils. Poldip2 (145 μl; 0.2 μM) and neutrophil membrane fractions (MF; 1.75 μl; 1.28 μM), containing the cytb558 (gp91phox/p22phox), were pre-incubated for 1 h with the cytb558/Poldip2 ratio of 1:20 (mol:mol). The MF and Polidp2 (alone) were treated similarly. The three samples were centrifuged at 190 000×g for 1h30. Pellet and supernatant were separated and analyzed by Western blot using antibodies raised against Nox2 (1:1500), p22phox (1:1500) and Poldip2 (1:1500). Experiments were performed at least three times with similar results.

Article Snippet: The membranes were incubated overnight at 4 ◦C with specific monoclonal or polyclonal antibodies: anti-gp91 (anti-gp91phox; 54.1; mouse monoclonal, dilution 1:1500; Santa Cruz), anti-p22 (44.1; mouse monoclonal; dilution 1:1500; Santa Cruz); anti-Poldip2 (ab181841; rabbit monoclonal; , dilution 1:1500; Abcam), anti-p67 (07-002; rabbit polyclonal; dilution 1:1000; Millipore); Anti-p47 (BD 610354; rabbit polyclonal; dilution 1:1500; Bioscience), anti-Rac1 (ARCO3; mouse monoclonal; dilution 1:500; Cytoskeleton), anti-NOX4 (ab109225; rabbit monoclonal; dilution 1:1500; Abcam).

Techniques: Purification, Membrane, Isolation, Incubation, Western Blot