phospho rela ser536  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: Transcranial microtesla magnetic fields suppress neuroinflammation and neuronal oxidative stress burden

doi: 10.1016/j.isci.2025.114425

Figure Lengend Snippet: MMT suppressed LPS-activated inflammatory signaling in mononuclear immune cells (A) Schematic of experimental protocol. hPBMCs were treated with vehicle control (DMSO) or LPS (100 ng/mL), with or without MMT, and cells were collected at 2 and 6 h for qPCR analysis. (B) mRNA levels for (i) Tnfα and (ii) Il-1β measured by qPCR for hPBMCs treated as in (A). Data from 2 independent donors. (C) (i) Schematic of LPS-induced NF-κB luciferase reporter, stably expressed in THP-1 and RAW264.7 cell lines, and experimental protocols for (ii) MMT co-treatment of THP-1 cells and (iii) MMT pre-treatment of THP-1 and Raw264.7 cells. (D) NF-κB luciferase activity in MMT-treated cells. MMT co- and pre-treated THP-1 cells and pre-treated RAW264.7 cells were treated for 6 h with vehicle (DMSO) or LPS (100 or 10 ng/mL), and luciferase activity was measured. (i) Representative data showing MMT suppression in co-treated THP-1 cells. Quantification of NF-κB suppression by MMT in (ii) co-treated and (iii) pre-treated THP-1 and RAW264.7 cells. (E) MMT reduced LPS-induced NF-κB p65 phosphorylation. THP-1 cells were treated with MMT or sham and stimulated with vehicle (DMSO) or LPS (100 ng/mL). Quantification of phosphorylation of p65 at Ser536 by immunofluorescence at 60 min post-LPS stimulation. (F) Schematic of LPS-sensitized pyroptosis in the THP-1 cell line. (G) Representative images of THP-1 cells under pyroptotic conditions. Maximum-intensity projections of z-stacks of sham and MMT-treated THP-1 cells primed with LPS (100 ng/mL) for 5 h, followed by 1 h treatment with ATP (5 mM). Hoechst (blue) labels nuclei, and propidium iodide (PI, red) labels cells with loss of membrane integrity. 63× objective; scale bars, 5 μm. (H) MMT reduced inflammatory cell death in THP-1 cells. Sham and MMT THP-1 cells were primed with LPS (100 ng/mL) for 5 h, treated with ATP (5 mM) for 1 h, and cell death was measured by counting PI-positive cells. Data are presented as mean ± SD. Statistical significance was determined by unpaired t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Samples were incubated overnight at 4 °C with rabbit anti-phospho-NF-κB p65 (Ser536) (R&D Systems, Cat# MAB72261), diluted in PBS with 1% FBS, followed by CoraLite Plus 488 goat anti-rabbit IgG (Proteintech, Cat# RGAR002) and Hoechst 33342 nuclear stain (Invitrogen, Cat# H3570) for 1 hour at RT, washed twice with PBS, and imaged on an EVOS 7000.

Techniques: Control, Luciferase, Stable Transfection, Activity Assay, Phospho-proteomics, Immunofluorescence, Membrane

Journal: Journal of Inflammation Research

Article Title: MRPL35 Attenuates Neonatal Parenteral Nutrition-Associated Cholestasis by Modulating the ROS/JNK/NF-κB Pathway

doi: 10.2147/JIR.S528466

Figure Lengend Snippet: Clinical Evaluation of the MRPL35/ROS/JNK/NF-κB Pathway in Neonates with PNAC. ( A ) Serum levels of TNF-α, IL-1β, IL-4, and IL-10 in neonates. *** P <0.001. ( B ) Apoptosis of PBMCs determined by flow cytometry. ** P <0.01. ( C ) Expression level of ROS in PBMCs was detected by immunofluorescence (×400). *** P <0.001. ( D ) Serum levels of MDA, Hydroxyl radical scavenging ability, SOD, and GSH-Px in neonates. ** P <0.01, *** P <0.001. ( E ) Expression of MRPL35 mRNA in PBMCs was detected by qRT-PCR. Values are means ± SD. * P <0.05. ( F ) Representative Western blot images of MRPL35, JNK, p-JNK, P65 and p-P65 in neonatal PBMCs. Values are means ± SD from three experiments. ** P <0.01. ( G ) Double immunofluorescence images for MRPL35 (red) + p-JNK (green) and MRPL35 (green) + p-P65 (red). DAPI staining (blue) indicates the nucleus. (scale bar =20 μm).

Article Snippet: The membranes were blocked and incubated with primary antibodies at the following dilutions: rabbit anti-MRPL35 (1:500, Affinity DF3668), rabbit anti-p65 (1:500, Novus NB100-2176), rabbit anti-phospho-p65 (1:500, Novus NB100-82088), mouse anti-JNK (1:500, Santa Cruz sc-7345), mouse anti-phospho-JNK (1:500, Santa Cruz sc-6254), and mouse anti-β-actin (1:1000, Santa Cruz sc-47778) overnight at 4°C.

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Quantitative RT-PCR, Western Blot, Staining

Journal: Journal of Inflammation Research

Article Title: MRPL35 Attenuates Neonatal Parenteral Nutrition-Associated Cholestasis by Modulating the ROS/JNK/NF-κB Pathway

doi: 10.2147/JIR.S528466

Figure Lengend Snippet: Assessment of the MRPL35/ROS/JNK/NF-κB Pathway in a PNAC Animal Model. ( A ) Levels of TNF-α and IL-1β in rat liver.*** P <0.001. ( B ) Hematoxylin and eosin (H&E) staining of rat liver tissue (×200). ( C ) Immunohistochemical images of TNF-α in rat liver tissue (×200). ( D ) Apoptosis of hepatocytes was determined by flow cytometry. *** P <0.001. ( E ) Expression level of ROS in hepatocytes was detected by immunofluorescence (×400). *** P <0.001. ( F ) Expression of MRPL35 mRNA in rat liver detected by qRT-PCR. *** P <0.001. ( G ) Double immunofluorescence images for MRPL35 (red) + p-JNK (green) and MRPL35 (green) + p-P65 (red). DAPI staining (blue) represents the nucleus (scale bar =20 μm).

Article Snippet: The membranes were blocked and incubated with primary antibodies at the following dilutions: rabbit anti-MRPL35 (1:500, Affinity DF3668), rabbit anti-p65 (1:500, Novus NB100-2176), rabbit anti-phospho-p65 (1:500, Novus NB100-82088), mouse anti-JNK (1:500, Santa Cruz sc-7345), mouse anti-phospho-JNK (1:500, Santa Cruz sc-6254), and mouse anti-β-actin (1:1000, Santa Cruz sc-47778) overnight at 4°C.

Techniques: Animal Model, Staining, Immunohistochemical staining, Flow Cytometry, Expressing, Immunofluorescence, Quantitative RT-PCR

Journal: Journal of Inflammation Research

Article Title: MRPL35 Attenuates Neonatal Parenteral Nutrition-Associated Cholestasis by Modulating the ROS/JNK/NF-κB Pathway

doi: 10.2147/JIR.S528466

Figure Lengend Snippet: Effects of MRPL35 Overexpression on a PNAC Animal Model. ( A ) Expression of MRPL35 mRNA in rat liver detected by qRT-PCR.*** P <0.001,** P <0.01. ( B ) Representative Western blot images of MRPL35, JNK, p-JNK, P65 and p-P65 in rat liver. Values are means ± SD from three experiments. *** P <0.001. ( C ) Serum levels of TNF-α and IL-1β in rats.*** P <0.001,* P <0.05. ( D ) Hematoxylin and eosin (H&E) staining of rat liver (×200). ( E ) Immunohistochemical image of TNF-α in rat liver (×200). ( F ) Apoptosis of hepatocytes determined by flow cytometry. *** P <0.001. ( G ) Expression level of ROS in hepatocytes detected by immunofluorescence (×400). *** P <0.001, ** P <0.01. ( H ) Serum levels of MDA, Hydroxyl radical scavenging ability, SOD, and GSH-pX. *** P <0.001, ** P <0.01, * P <0.05.

Article Snippet: The membranes were blocked and incubated with primary antibodies at the following dilutions: rabbit anti-MRPL35 (1:500, Affinity DF3668), rabbit anti-p65 (1:500, Novus NB100-2176), rabbit anti-phospho-p65 (1:500, Novus NB100-82088), mouse anti-JNK (1:500, Santa Cruz sc-7345), mouse anti-phospho-JNK (1:500, Santa Cruz sc-6254), and mouse anti-β-actin (1:1000, Santa Cruz sc-47778) overnight at 4°C.

Techniques: Over Expression, Animal Model, Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemical staining, Flow Cytometry, Immunofluorescence

Journal: Frontiers in Oncology

Article Title: Targeting RAB7 in human B lymphoma by a small molecule inhibitor arrests tumor cell growth

doi: 10.3389/fonc.2025.1616519

Figure Lengend Snippet: RAB7 expression in human tonsil B cells. (A) Representative flow cytometry plots showing the gating strategy used to identify CD19 - non-B cells, CD19 + IgD + and CD19 + IgD - B cell subsets from human tonsil samples (left) and the levels of RAB7, NF-κB RELA subunit and phosphorylated NF-κB RELA. (B) Quantification of the mean fluorescence intensity (MFI) of RAB7, NF-κB RELA, phosphorylated NF-κB RELA and the ratio of phosphorylated NF-κB over total NF-κB in different immune cell and B cell populations (n=3; mean and s.d.). * p < 0.05, ** p < 0.01; one-way ANOVA.

Article Snippet: The cells were fixed and stained intracellularly with fluorophore-labeled mAb to RAB7 (Abcam; cat# ab198337), total RELA (CST; cat# 9609) and phosphorylated RELA (CST; cat# 4886), following the instructions of the BD intracellular staining kit (cat# 554714).

Techniques: Expressing, Flow Cytometry, Fluorescence