Journal: Heliyon

Article Title: Identification of signaling networks associated with lactate modulation of macrophages and dendritic cells

doi: 10.1016/j.heliyon.2025.e42098

Figure Lengend Snippet: Effect of sLA on signaling proteins of interest in DCs and MΦs. ( A ) Schematic experiment was depicted. DCs and MΦs were exposed to either 50 mM sLA or control media, and incubated for 48 h. For Western blot analysis, proteins were separated by size and then transferred to the membrane. Protein detection was done using primary antibodies against several proteins including p-STAT3, p-ERK1/2, p-p38 MAPK, p-STAT1, p-STAT2, p-GSK-3β and β-actin. For flow cytometry, DCs cell suspensions were stained with fluorescently tagged anti-CD11c, p-STAT3, p-ERK and p-p38 MAPK. MΦs were stained with fluorescently tagged anti-F4/80, p-STAT1 and p-GSK-3β. The analysis of the phosphorylated protein expression was performed using a flow cytometer. ( B ), ( D ) Western blot results with represented blots showed the effect of sLA on the levels of p-STAT3, p-ERK1/2 and p-p38 MAPK for DCs. For MΦs, p-STAT1, p-STAT2, p-GSK-3β were shown for the effect of sLA. ( C ), ( E ) Flow cytometric assessment revealed the levels of phosphorylated proteins expression in DCs and MΦs after sLA treatment. The data are demonstrated as the mean ± S.E. from at least three independent experiments, with statistically significant differences calculated by comparing each treatment group to the control. The following symbols indicate the corresponding p-values: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 as determined by a two-tailed one-sample t -test.

Article Snippet: The membrane was incubated with each primary antibody targeting phosphorylated STAT1 (p-STAT1, Tyr701, D4A7, Cell signaling technology (CST) #7649), p-STAT2 (Tyr690, CST #4441), p-STAT3 (Ser727, CST #9134), p-SAPK/JNK (Thr183/Tyr185, 81E11, CST #4668), p-SAPK/JNK (Thr183/Tyr185, 98F2, CST #4671), p-NF-κB p65 (Ser536, 93H1, CST #3033), β-Catenin (Ser45, D2U8Y, CST #19807), p-Akt (Ser473, CST #9271), p-Akt (Thr308, D25E6, CST #13038), p-Akt (Ser473, D9E, CST #4060), Akt (pan) (C67E7, CST #4691), p-PTEN (Ser380, CST #9551), p-GSK-3β (Ser9, D85E12, CST #5558), p-c-Raf (Ser259, CST #9421), p-PDK1 (Ser241, C49H2, CST #3438), p-p44/42 MAPK (Erk1/2, Thr202/Tyr204, D13.14.4E, CST #4370), p-p38 MAPK (Thr180/Tyr182, D3F9, CST #4511), and β-actin (CST #4967).

Techniques: Control, Incubation, Western Blot, Membrane, Flow Cytometry, Staining, Expressing, Two Tailed Test

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Myeloid Deletion of Cdc42 Protects Liver From Hepatic Ischemia-Reperfusion Injury via Inhibiting Macrophage-Mediated Inflammation in Mice

doi: 10.1016/j.jcmgh.2024.01.023

Figure Lengend Snippet: Myeloid deletion of Cdc42 enhances the M2-type differentiation of macrophages via regulating STATs signaling. THP1 cells were pretreated with PMA (100 ng/mL) and ML141 (10 μM) for differentiation and then stimulated with LPS (100 ng/mL) and IFN-γ (20 ng/mL) for 1 hour for M1 induction, or IL4 and IL13 (both at 40 ng/mL) for 48 hours for M2 induction. The images ( A ) and quantitative results ( C–F ) of the phosphorylation of STAT1, STAT3, and STAT6 and expressions of SOCS3 were detected by Western blot analysis in THP1 cells, n = 3. The images ( B ) and quantitative results ( G–J ) of the phosphorylation of STAT1, STAT3, and STAT6 and expressions of SOCS3 were measured by Western blot analysis in BMDMs stimulated with LPS (100 ng/mL) for 3 hours or IL4 (40 ng/mL) for 48 hours, respectively, n = 3. n. s., no significance; ∗ P < .05; ∗∗∗ P < .001.

Article Snippet: The proteins were extracted and resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare), which will be blocked with 5% BSA or skimmed milk in Tris-buffered saline containing 0.1% Tween 20 and probed with antibodies against iNOS (Proteintech, 22226-1-AP), ARG1 (Proteintech, 66129-1-AP), IL10 (proteintech,60269-1-Ig), IL6 (CST, 12912S), SOCS3 (Proteintech, 14025-1-AP), STAT Antibody Sampler Kit (CST, 9939T), Phospho-STAT Antibody Sampler (CST, 9914T: Phospho-STAT1 Tyr701 and Phospho-STAT3 Tyr705, Phospho-STAT6 Tyr641), respectively.

Techniques: Western Blot