phosphor p38  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: Macrophage migration inhibitory factor of zoonotic Giardia duodenalis triggers TLR4-MAPK/AKT/NLRP3 pathways to regulate inflammatory response

doi: 10.1016/j.isci.2025.114404

Figure Lengend Snippet: rGdMIF activates MAPK, AKT, and NF-κB signaling pathway in mouse PMϕs (A and E) PMϕs were stimulated with rGdMIF (1 μg/mL) at different times, the expression of total and phosphorylated proteins of p38, ERK, AKT, p65, and IκB were detected by western blot. (B–D, F, and G) The expression level of protein was analyzed by ImageJ. (H and L) PMϕs were stimulated with different concentration rGdMIF for 2 h, the expression of total and phosphorylated proteins of p38, ERK, AKT, p65, and IκB were detected by western blot. (I–K, M, and N) The expression level of protein was analyzed by ImageJ. Data are presented as mean ± SD from three independent experiments ( n = 3), statistical significance was assessed using one-way ANOVA. n.s. ( p > 0.05) indicates not significant, and ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Phosphor-p38 ( Thr180/Tyr182 ) , Cell Signaling Technology , Cat#:#9211; RRID: AB_331641.

Techniques: Expressing, Western Blot, Concentration Assay

Journal: iScience

Article Title: Macrophage migration inhibitory factor of zoonotic Giardia duodenalis triggers TLR4-MAPK/AKT/NLRP3 pathways to regulate inflammatory response

doi: 10.1016/j.isci.2025.114404

Figure Lengend Snippet: Roles of TLR4 receptor in rGdMIF triggered MAPK, AKT, and NF-κB signaling pathway WT, TLR2 −/− and TLR4 −/− PMϕs were stimulated with rGdMIF (1 μg/mL) for 2 h, respectively. (A and E) The expression of total and phosphorylated p38, ERK, AKT, p65, and IκB were detected by western blot. (B–D, F, and G) The expression level of protein was analyzed by ImageJ. Data are presented as mean ± SD from three independent experiments ( n = 3), statistical significance was assessed using one-way ANOVA. n.s. ( p > 0.05) indicates not significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Phosphor-p38 ( Thr180/Tyr182 ) , Cell Signaling Technology , Cat#:#9211; RRID: AB_331641.

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: Macrophage migration inhibitory factor of zoonotic Giardia duodenalis triggers TLR4-MAPK/AKT/NLRP3 pathways to regulate inflammatory response

doi: 10.1016/j.isci.2025.114404

Figure Lengend Snippet: rGdMIF regulated inflammatory cytokines secretion through the p38, ERK, and AKT signaling pathways WT mouse PMϕs were pretreated with SB203580 (30 μM), PD98059 (40 μM), or MK-2206 2HCl (5 μM) and stimulated with rGdMIF (1 μg/mL). (A–C) The phosphorylation levels of p38, ERK, and AKT proteins were detected by western blot, and the expression levels of proteins were analyzed by ImageJ. Data are displayed as mean ± SD, statistical significance was assessed using unpaired Student’s t test. (D–H) The productions of cytokines were measured by ELISA. Data are presented as mean ± SD from three independent experiments ( n = 3), statistical significance was assessed using one-way ANOVA. n.s. ( p > 0.05) indicates not significant, and ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Phosphor-p38 ( Thr180/Tyr182 ) , Cell Signaling Technology , Cat#:#9211; RRID: AB_331641.

Techniques: Protein-Protein interactions, Phospho-proteomics, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Macrophage migration inhibitory factor of zoonotic Giardia duodenalis triggers TLR4-MAPK/AKT/NLRP3 pathways to regulate inflammatory response

doi: 10.1016/j.isci.2025.114404

Figure Lengend Snippet: rGdMIF activates MAPK, AKT, and NF-κB signaling pathway in mouse PMϕs (A and E) PMϕs were stimulated with rGdMIF (1 μg/mL) at different times, the expression of total and phosphorylated proteins of p38, ERK, AKT, p65, and IκB were detected by western blot. (B–D, F, and G) The expression level of protein was analyzed by ImageJ. (H and L) PMϕs were stimulated with different concentration rGdMIF for 2 h, the expression of total and phosphorylated proteins of p38, ERK, AKT, p65, and IκB were detected by western blot. (I–K, M, and N) The expression level of protein was analyzed by ImageJ. Data are presented as mean ± SD from three independent experiments ( n = 3), statistical significance was assessed using one-way ANOVA. n.s. ( p > 0.05) indicates not significant, and ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Membranes were blocked with a protein-free rapid blocking solution (EpiZyme, Shanghai, China) for 30 min, membranes were incubated with primary antibodies (anti-Phosphor-p38 ( Thr180/Tyr182 ), anti-p38, anti-phospho-ERK ( Thr202/Tyr204 ), anti-ERK, anti-Phospho-AKT ( Ser473 ), anti-AKT, anti-NF-κB p65, anti-Phospho-p65 (Ser536), anti-Phospho-IκBα (Ser32), anti-IκBα (all rabbit), and mouse anti-GAPDH, diluted at 1:1,000 in 5% BSA) (Cell Signaling Technology, Danvers, USA) at 4°C for overnight, washed 3 times with Tris-Buffered Saline with Tween 20 (TBST) (Servicebio, Wuhan, China), incubated with HRP-conjugated Goat Anti-Mouse IgG(H + L) or HRP-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech, Wuhan, China) at RT for 1 h, then washed with TBST.

Techniques: Expressing, Western Blot, Concentration Assay

Journal: iScience

Article Title: Macrophage migration inhibitory factor of zoonotic Giardia duodenalis triggers TLR4-MAPK/AKT/NLRP3 pathways to regulate inflammatory response

doi: 10.1016/j.isci.2025.114404

Figure Lengend Snippet: Roles of TLR4 receptor in rGdMIF triggered MAPK, AKT, and NF-κB signaling pathway WT, TLR2 −/− and TLR4 −/− PMϕs were stimulated with rGdMIF (1 μg/mL) for 2 h, respectively. (A and E) The expression of total and phosphorylated p38, ERK, AKT, p65, and IκB were detected by western blot. (B–D, F, and G) The expression level of protein was analyzed by ImageJ. Data are presented as mean ± SD from three independent experiments ( n = 3), statistical significance was assessed using one-way ANOVA. n.s. ( p > 0.05) indicates not significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Membranes were blocked with a protein-free rapid blocking solution (EpiZyme, Shanghai, China) for 30 min, membranes were incubated with primary antibodies (anti-Phosphor-p38 ( Thr180/Tyr182 ), anti-p38, anti-phospho-ERK ( Thr202/Tyr204 ), anti-ERK, anti-Phospho-AKT ( Ser473 ), anti-AKT, anti-NF-κB p65, anti-Phospho-p65 (Ser536), anti-Phospho-IκBα (Ser32), anti-IκBα (all rabbit), and mouse anti-GAPDH, diluted at 1:1,000 in 5% BSA) (Cell Signaling Technology, Danvers, USA) at 4°C for overnight, washed 3 times with Tris-Buffered Saline with Tween 20 (TBST) (Servicebio, Wuhan, China), incubated with HRP-conjugated Goat Anti-Mouse IgG(H + L) or HRP-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech, Wuhan, China) at RT for 1 h, then washed with TBST.

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: Macrophage migration inhibitory factor of zoonotic Giardia duodenalis triggers TLR4-MAPK/AKT/NLRP3 pathways to regulate inflammatory response

doi: 10.1016/j.isci.2025.114404

Figure Lengend Snippet: rGdMIF regulated inflammatory cytokines secretion through the p38, ERK, and AKT signaling pathways WT mouse PMϕs were pretreated with SB203580 (30 μM), PD98059 (40 μM), or MK-2206 2HCl (5 μM) and stimulated with rGdMIF (1 μg/mL). (A–C) The phosphorylation levels of p38, ERK, and AKT proteins were detected by western blot, and the expression levels of proteins were analyzed by ImageJ. Data are displayed as mean ± SD, statistical significance was assessed using unpaired Student’s t test. (D–H) The productions of cytokines were measured by ELISA. Data are presented as mean ± SD from three independent experiments ( n = 3), statistical significance was assessed using one-way ANOVA. n.s. ( p > 0.05) indicates not significant, and ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Membranes were blocked with a protein-free rapid blocking solution (EpiZyme, Shanghai, China) for 30 min, membranes were incubated with primary antibodies (anti-Phosphor-p38 ( Thr180/Tyr182 ), anti-p38, anti-phospho-ERK ( Thr202/Tyr204 ), anti-ERK, anti-Phospho-AKT ( Ser473 ), anti-AKT, anti-NF-κB p65, anti-Phospho-p65 (Ser536), anti-Phospho-IκBα (Ser32), anti-IκBα (all rabbit), and mouse anti-GAPDH, diluted at 1:1,000 in 5% BSA) (Cell Signaling Technology, Danvers, USA) at 4°C for overnight, washed 3 times with Tris-Buffered Saline with Tween 20 (TBST) (Servicebio, Wuhan, China), incubated with HRP-conjugated Goat Anti-Mouse IgG(H + L) or HRP-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech, Wuhan, China) at RT for 1 h, then washed with TBST.

Techniques: Protein-Protein interactions, Phospho-proteomics, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Parasites & Vectors

Article Title: Interactions between bacterial vaginosis-associated microbiota and Trichomonas vaginalis modulate parasite-induced pathogenicity and host immune responses

doi: 10.1186/s13071-025-06996-5

Figure Lengend Snippet: Tv pretreated with specific bacterial species enhances the activation of PI3K and MAPK signaling pathways in Ect1 cells. Tv was co-incubated with E. coli , L. crispatus (LCR), P. bivia , and L. iners at a ratio of 1:10 for 6 h. After co-incubation, Ect1 cells were exposed to Tv for 30, 60, and 120 min, and Ect1 cell lysates were subsequently analyzed by western blotting using antibodies specific for phosphorylated PI3K ( p -PI3K) a , ERK ( p -ERK) b , and p38 MAPK ( p -p38 MAPK) c . Tv cultivation alone (Tv only) served as the control. β -actin was used as a loading control for western blotting. Band intensities of each target protein were quantified using ImageJ software and normalized to the corresponding β -actin band from the same lane. Quantitative data are presented as mean ± SD from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The following primary antibodies were used for immunoblotting: antibodies against phosphor-PI3K, phosphor-ERK, phosphor-p38 MAPK, E -cadherin, Snail (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA), and β -Actin (1:2000; Cell Signaling Technology Inc., Danvers, MA, USA).

Techniques: Activation Assay, Protein-Protein interactions, Incubation, Western Blot, Control, Software

Journal: Parasites & Vectors

Article Title: Interactions between bacterial vaginosis-associated microbiota and Trichomonas vaginalis modulate parasite-induced pathogenicity and host immune responses

doi: 10.1186/s13071-025-06996-5

Figure Lengend Snippet: Schematic model illustrating the crosstalk between Tv and various vaginal bacterial species in modulating parasite pathogenesis and host immune responses. Specific BVB, such as P. bivia , acts as pathobionts for Tv, enhancing Tv adherence to Ect1 cells and exacerbating Ect1 cytotoxicity. Additionally, Tv pretread with P. bivia activates the PI3K, ERK, and p38 MAPK signaling pathways, as well as EMT in Ect1 cells, leading to increased secretion of the pro-inflammatory cytokines, which may contribute to disease progression in the female reproductive system

Article Snippet: The following primary antibodies were used for immunoblotting: antibodies against phosphor-PI3K, phosphor-ERK, phosphor-p38 MAPK, E -cadherin, Snail (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA), and β -Actin (1:2000; Cell Signaling Technology Inc., Danvers, MA, USA).

Techniques: Protein-Protein interactions, Biomarker Discovery