phosphorylated nf kb p65  (Novus Biologicals)

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a , b iMac were cultured in 6-well plates (3 × 10 6 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, STAT1, STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a – c , one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. * p < 0.05, *** p < 0.001, and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Phospho-proteomics, Western Blot, Software

Journal: Pathogens

Article Title: Differential TLR-ERK1/2 Activity Promotes Viral ssRNA and dsRNA Mimic-Induced Dysregulated Immunity in Macrophages

doi: 10.3390/pathogens13121033

Figure Lengend Snippet: RNA mimic stimulation induced differential phosphorylation of MAPK and NF-kB signaling. Murine bone marrow macrophages were treated with ssRNA mimic (R848) and dsRNA mimic (PIC), each with 10 ug/mL, for 10, 30, and 60 min. Cell lysates were collected, and Western blot was performed using mAbs against phosphorylated MAPK (p-ERK1/2 (phospho-ERK1/2), p-P38 (phospho-P38), and p-JNK (phospho-JNK) and NF-kB (p-P65 (phospho-P65)). and their total proteins were denoted as T-ERK, T-P38, T-JNK, and T-P65, respectively ( A ), and relative quantification of protein levels were measured by densitometry analysis ( B ). Data are representative of three independent experiments ( A ) or pooled from three independent experiments ( B ). Statistical significance was determined using one-way ANOVA with * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: The primary antibodies used included anti-phosphorylated p44/42 MAPK (Cell signaling # 9101, 1:1000), anti-p44/42 MAPK (Cell signaling # 4695, 1:1000), anti-phosphorylated p38 MAPK (Cell signaling # 4511, 1:1000), anti-p38 MAPK (Cell signaling # 8690, 1:1000), anti-phosphorylated SAPK/JNK (Cell signaling # 9255, 1:1000), anti-SAPK/JNK (Cell signaling # 9252, 1:1000), anti-phosphorylated NF-kB p65 (Cell signaling # 3033, 1:1000), anti-NF-kB p65 (Cell signaling # 8242, 1:1000), and β-actin (Invitrogen # MA5-15739).

Techniques: Phospho-proteomics, Western Blot, Quantitative Proteomics

Journal: Pathogens

Article Title: Differential TLR-ERK1/2 Activity Promotes Viral ssRNA and dsRNA Mimic-Induced Dysregulated Immunity in Macrophages

doi: 10.3390/pathogens13121033

Figure Lengend Snippet: Effect of blocking MAPK and NF-kB signaling in viral RNA mimic-induced inflammatory and antiviral cytokine response. Murine BMDMs were treated with specific inhibitors (5 µM) of ERK1/2 ( i ), p38 ( ii ), NF-kB ( iii ), and JNK ( iv ) pathways for 2 h followed by stimulation with R848 and or Poly I:C, each with 5–10 ug/mL. Western blot showing the inhibitory effect of Trametinib, Losmapimod, BAY, and JNKi on phospho-ERK (p-ERK), phospho-P38 (p-P38), phospho-P65(p-P65), and phospho-JNK(p-JNK), respectively, and their total proteins, denoted as T-ERK, T-P38, T-P65, and T-JNK, respectively. ( A ) Protein levels of cytokines and interferons ( B , C ) were assessed at 8 and 24 h post stimulation. Data are representative of 2–3 independent experiments ( B , C ). Statistical significance was determined using one-way ANOVA with ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: The primary antibodies used included anti-phosphorylated p44/42 MAPK (Cell signaling # 9101, 1:1000), anti-p44/42 MAPK (Cell signaling # 4695, 1:1000), anti-phosphorylated p38 MAPK (Cell signaling # 4511, 1:1000), anti-p38 MAPK (Cell signaling # 8690, 1:1000), anti-phosphorylated SAPK/JNK (Cell signaling # 9255, 1:1000), anti-SAPK/JNK (Cell signaling # 9252, 1:1000), anti-phosphorylated NF-kB p65 (Cell signaling # 3033, 1:1000), anti-NF-kB p65 (Cell signaling # 8242, 1:1000), and β-actin (Invitrogen # MA5-15739).

Techniques: Blocking Assay, Western Blot