Journal: Journal of Biological Chemistry

Article Title: Atypical Protein Kinase Cι Plays a Critical Role in Human Lung Cancer Cell Growth and Tumorigenicity

doi: 10.1074/jbc.m505402200

Figure Lengend Snippet: FIG. 2. PKC does not regulate NFB signaling in A549 cells. A, immunoblot analysis of A549 cell transfectants for cIAP2, Bcl-XL, and actin. B, analysis of A549 cell transfectants for PARP and cleaved PARP. HeLa cells treated with taxol for either 24 or 48 h served as a positive control for cleaved PARP. C, A549/pBabe and A549/kdPKC cells were treated with 50 g/ml TNF for the indicated times and cell lysates subjected to immunoblot analysis for detection of IB, phospho-(Ser32)-IB, and actin. D, A549/pBabe and A549/kdPKC cells were treated with 50 g/ml TNF for the indicated times and cell lysates subjected to immunoblot analysis for detection of NFB p65, phospho-(Ser536)-NFB, p65, and actin. E, transcriptional activity of an NFB-luciferase reporter in A549/pBabe and A549/kdPKC cells in the presence and absence of TNF. Data represent the mean S.E. of three independent determinations.

Article Snippet: This paper is available on line at http://www.jbc.org 31109 at K U N G L IG A T E K N ISK A H O G SK O L A N on M arch 8, 2015 http://w w w .jbc.org/ D ow nloaded from (Sigma, catalog number A8592); Rac1, 1:3000 (BD Transduction Laboratories, catalog number 610651); cIAP2, 1:500 (Santa Cruz Biotechnology, catalog number sc-7944); Bcl-XL, 1:1,000 (Cell Signaling Technologies, catalog number 2762); PARP/cleaved PARP, 1:1,000 (Cell Signaling Technologies, catalog number 9542); I B , 1:1,000 (Cell Signaling Technologies, catalog number 9242); phospho-(Ser32)-I B , 1:1,000 (Cell Signaling Technologies, catalog number 9241); NF B p65, 1:1,000 (Cell Signaling Technologies, catalog number 3034); phospho(Ser536)-NF B p65, 1:1,000 (Cell Signaling Technologies, catalog number 3031); Mek, phospho-(Ser217/221)-Mek, and phospho-(Ser298)Mek, each at 1:1,000 (Cell Signaling Technologies, catalog numbers 9122, 9121, and 9128, respectively), Erk and phospho-(Thr202/Tyr204)Erk, each at 1:1,000 (Cell Signaling Technologies, catalog numbers 9102 and 9101, respectively); CD31, 1:1,000 (or Pecam-1; Santa Cruz Biotechnology, catalog number sc-1506); BrdUrd, 1:100 (DAKO, catalog number M0744).

Techniques: Western Blot, Positive Control, Activity Assay, Luciferase

Journal: Scientific Reports

Article Title: p38α MAPK regulates proliferation and differentiation of osteoclast progenitors and bone remodeling in an aging-dependent manner

doi: 10.1038/srep45964

Figure Lengend Snippet: ( A ) TRAP staining showed that p38α deficiency promoted osteoclast differentiation in low cell density culture but slightly inhibited osteoclast differentiation in high cell density cultures. Monocytes were isolated from LysM-Cre; p38α f/f and control mice, plated at different densities, and cultured in the presence of RANKL and M-CSF. After 7 days, the cultures were stained for TRAP (left panels). Right panels: quantitation data. Scale bar, 200 μm. N = 3. ( B ) Quantitative PCR results showed that p38α deficiency promoted the expression of genes required for osteoclast differentiation at low cell densities. Monocytes described in Fig. 2A (low cell density) were collected and used to isolate total RNA, which was used to run quantitative PCR assays. N = 3. ( C ) Quantitative PCR results showed that p38α deficiency slightly inhibited the expression of genes required for osteoclast differentiation at high cell densities. ( D ) Western blot results showed that p38α deficient monocytes cultures at low densities exhibited an increase in activation of Tak1 and the protein levels of NF-κB isoforms p50/52 and p65. Scale bar, 100 μm. ( E ) Inhibition of Tak1 down-regulated NF-κB levels and suppressed osteoclast differentiation. Left panel: western blot showed that Tak1 inhibitor down-regulated NF-κB levels. Middle panel: Tak1 inhibitor suppressed osteoclast differentiation. Right panel: Quantitation data. N = 3. ( F ) Western blot results revealed that p38α deficient monocyte cultures at high densities showed a decrease in c-Fos protein levels. ( G ) p38α deficiency did not affect the resorbing activity of osteoclasts on dentine slices. WT and p38 deficient monocytes were induced to differentiate into osteoclasts by M-CSF and RANKL for 2 days and then counted and the same numbers of cells were plated onto dentine slices. After 7 days, the dentine slices were sonicated and stained with Gill’s hematoxylin. Right panel: quantitation data. Scale bar, 200 μm. N = 3. For all results in Fig. 2, P-values are based on Student’s t-test. *p < 0.05, **p < 0.01 when the value of mutant mice or cells was compared to that of control mice or cells, or the drugs-treated group compared to control group.

Article Snippet: The specific antibodies used in this study were: NF-κB p65 (Cell Signaling, 4767, Boston, MA), NF-κB p50/52 (Santa Cruz, sc-8414, California), p38α MAPK (Cell Signaling, 9212, Boston, MA), p38β (Cell Signaling, 2339, Boston, MA), p-p38MAPK (T180/182) (Cell Signaling, 9211, Boston, MA), p53 (c12) (Cell Signaling, 2524, Boston, MA), Tak1 (Cell Signaling, 4505, Boston, MA), p-Tak1(T184/187) (Cell Signaling, 4531, Boston, MA), Creb (Upstate, 05767, Boston, MA), p-Creb (Upstate, 6519, Boston, MA), ERK (Cell Signaling, 9107, Boston, MA), p-ERK (Cell Signaling, 9106s, Boston, MA), cFos (Calbiochem, pc38, Darmstadt, Germany), p-cFos (Santa Cruz, sc-81485, California), and β-ACTIN (Santa Cruz, sc-81178, California).

Techniques: Staining, Isolation, Control, Cell Culture, Quantitation Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Activation Assay, Inhibition, Activity Assay, Sonication, Mutagenesis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SCRIPT Regulates IL-10 Production in Human Dendritic Cells by Modulating NF-κBp65 Activation.

doi: 10.4049/jimmunol.1402924

Figure Lengend Snippet: FIGURE 2. TLR-induced IL-10 produc- tion in SCRIPT-KD-DCs depends on NF- kBp65. (A) Immature SCRIPT-KD-DCs and Ctrl-DCs were incubated with BAY11- 7082 for 1 h, followed by a 24-h incubation with R848. Supernatants were harvested and assayed for the effect of NF-kB inhi- bition on IL-10 production. Statistics: Stu- dent two-tailed paired t test, n = 6; error bars = SEM. (B) Immature SCRIPT-KD- DCs and Ctrl-DCs were stimulated for 1 h with R848. Nuclear lysates were isolated and allowed to react with the consensus sequence for NF-kB on a precoated 96-well plate. Different subunits of NF-kB were detected using mAbs for the protein of in- terest, together with a colorimetric readout. Statistics: one-way ANOVA with a Bonfer- roni posttest; n = 4–5; error bars = SEM. *p , 0.05, **p , 0.01.

Article Snippet: Cells were blocked and stained in PBS with 1% BSA, 0.05% NaN3, and 2% human serum, using 1:100 diluted acetyl-K310 NF-kBp65 Ab (catalog no. 3045; Cell Signaling Technology) or isotype control (rabbit IgG; Jackson Immunoresearch).

Techniques: Incubation, Two Tailed Test, Isolation, Sequencing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SCRIPT Regulates IL-10 Production in Human Dendritic Cells by Modulating NF-κBp65 Activation.

doi: 10.4049/jimmunol.1402924

Figure Lengend Snippet: FIGURE 3. DC-SCRIPT knockdown increases phosphorylation of NF- kBp65. SCRIPT-KD-DCs or Ctrl-DCs were stimulated with R848 for 0–120 min. Protein lysates were assayed by Western blot for phosphorylated NF-kBp65, total NF-kBp65, and actin as loading control. (A) Represen- tative donor displaying that DC-SCRIPT knockdown induces a higher phosphorylated NF-kBp65. (B and C) p-NF-kBp65 and total NF-kBp65 were quantified for a range of donors. Statistics: Ctrl-DCs were compared with SCRIPT-KD-DCs for each time point, using the Student two-tailed t test (n = 3–4). Error bars = SEM. *p , 0.05.

Article Snippet: Cells were blocked and stained in PBS with 1% BSA, 0.05% NaN3, and 2% human serum, using 1:100 diluted acetyl-K310 NF-kBp65 Ab (catalog no. 3045; Cell Signaling Technology) or isotype control (rabbit IgG; Jackson Immunoresearch).

Techniques: Knockdown, Phospho-proteomics, Western Blot, Control, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SCRIPT Regulates IL-10 Production in Human Dendritic Cells by Modulating NF-κBp65 Activation.

doi: 10.4049/jimmunol.1402924

Figure Lengend Snippet: FIGURE 4. DC-SCRIPT knockdown increases acetylation of NF-kBp65. Ctrl- DCs or SCRIPT-KD-DCs (SC) were stim- ulated for 0–60 min with R848, followed by intracellular acetyl NF-kBp65 (K310) FACS staining. (A and B) Overlays between unstimulated and 30-min R848-stimulated moDCs. (C) Positive control: LPS + anti– DC-SIGN Ab (DCN46). (D and E) moDCs were pretreated for 1 h with the HATi AA or vehicle control before R848 stimulation to assay the effect of HATs on the observed acetylation. The line going through (A)–(E) indicates the peak of unstimulated SCRIPT- KD-DCs. (F) Summary of the relative fluo- rescence intensity (FI) for siRNA-treated moDCs stimulated with R848. Values are normalized to the mean FI for unstimulated Ctrl-DCs. Statistics: ANOVA with a Bon- ferroni posttest, n = 3–4. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Cells were blocked and stained in PBS with 1% BSA, 0.05% NaN3, and 2% human serum, using 1:100 diluted acetyl-K310 NF-kBp65 Ab (catalog no. 3045; Cell Signaling Technology) or isotype control (rabbit IgG; Jackson Immunoresearch).

Techniques: Knockdown, Staining, Positive Control, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SCRIPT Regulates IL-10 Production in Human Dendritic Cells by Modulating NF-κBp65 Activation.

doi: 10.4049/jimmunol.1402924

Figure Lengend Snippet: FIGURE 6. DC-SCRIPT knockdown increases NF-kBp65 binding to the IL10 enhancer. SCRIPT-KD-DCs and Ctrl-DCs were stimulated for 60 min with R848 and ChIPed with an NF-kBp65 Ab or isotype control (IgG). The ChIPed product and input chromatin for the ChIP was assayed for IL10 enhancer binding by qPCR. The recovery was calculated by relating the ChIPed product to the input. Statistics: ANOVA with a Bonferroni posttest; n = 4; error bars = SEM. *p , 0.05, **p , 0.01.

Article Snippet: Cells were blocked and stained in PBS with 1% BSA, 0.05% NaN3, and 2% human serum, using 1:100 diluted acetyl-K310 NF-kBp65 Ab (catalog no. 3045; Cell Signaling Technology) or isotype control (rabbit IgG; Jackson Immunoresearch).

Techniques: Knockdown, Binding Assay, Control

Journal: The Journal of Physiology

Article Title: Altered anabolic signalling and reduced stimulation of myofibrillar protein synthesis after feeding and resistance exercise in people with obesity

doi: 10.1113/JP276210

Figure Lengend Snippet: Muscle protein content for TLR4 (A), MyD88 (B), total protein (C) and phosphorylation of NF‐κB at Ser468 (D) at basal and after the ingestion of pork (n = 9 per group) in both the non‐exercised (CON) and exercised (EX) leg. Immediately prior to pork ingestion, participants performed a bout of unilateral leg extension exercise. Data are the mean ± SEM. * P < 0.05 vs. basal, #P < 0.05 vs. 120 min, †P < 0.05 vs. CON leg, ‡P < 0.05 vs. NW.

Article Snippet: Phosphorylation status and total protein content of mammalian target of rapamycin complex 1 at Ser2448 (mTORC1: 1° dilution 1:500, catalogue number 2971; Cell Signaling Technology, Beverly, MA USA), 70 kDa S6 protein kinase 1 at Thr389 (p70S6K: 1° dilution 1:500, catalogue number 9205; Cell Signaling Technology), eukaryotic initiation factor 4E binding protein 1 at Thr37/46 (4E‐BP1: 1° dilution 1:1000, catalogue number 9459; Cell Signaling Technology) and nuclear factor‐kappa B (NF‐κB) at Ser468 (NF‐κB: 1° dilution 1:1000, catalogue number 3039; Cell Signaling Technology) were determined using antibodies purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Silence of lncRNA MIAT protects ATDC5 cells against lipopolysaccharides challenge via up-regulating miR-132.

doi: 10.1080/21691401.2019.1626410

Figure Lengend Snippet: Figure 4. Suppressive effect of MIAT silence on the activation of NF-jB and JNK pathways via miR-132. ATDC5 cells were co-transfected with sh-MIAT and miR-132 inhibitor and then subjected to 6 lg/mL LPS. The phosphor/total (p/t) levels of (A,B) p65, IjBa and (C,D) JNK were measured using western blot (n ¼ 3). p < .05 (ANOVA combined with Duncan post-hoc test).

Article Snippet: Anti-caspase-3 (orb378617), anti-cleavedcaspase-3 (orb106556), anti-caspase-9 (orb135175), anticleaved-caspase-9 (orb227889), anti-PARP (orb526607), anti-cleaved-PARP (orb106557), anti-IL-6 (orb6210), anti-IL-8 (orb39299), anti-TNF-a (orb475251), anti-MCP-1 (orb97456), anti-p65 (orb344389), anti-p-p65 (orb14753), anti-IjBa (orb338946), anti-p-IjBa (orb99312), anti-JNK (orb38050), anti-p-JNK (orb184488), and anti-b-actin (orb86987) all from Biorbyt (San Francisco, CA).

Techniques: Activation Assay, Transfection, Western Blot

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: A Topical Chinese Herbal Alleviates Psoriasis by Regulating Keratinocytes Pyroptosis Through Inhibition of NLRP3 Inflammasome Activation

doi: 10.2147/CCID.S559712

Figure Lengend Snippet: ( A – C ) mRNA levels of NLRP3, Caspase-1, GSDMD in NHEK cells, n = 3. ( D – I ) Protein levels of NF-κB, P65, NLRP3, caspase-1, GSDMD in NHEK cells, n = 3.*P < 0.05, **P < 0.01, ***P < 0.001 compared with Control; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with Model; & P < 0.05, && P < 0.01, &&& P < 0.001 compared with QS; ^P < 0.05, ^^P < 0.01 compared with Mcc950.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: NF-κB p65 Monoclonal antibody (66535-1-Ig, Proteintech, China), NLRP3 Monoclonal antibody (68102-1-Ig-100, Proteintech, China), Caspase-1 antibody (sc-56036, Santa Cruz, USA), and GSDMD Polyclonal antibody (20770-1-AP, Proteintech, China).

Techniques: Control

Journal: Cell stem cell

Article Title: PGC-1α Controls Skeletal Stem Cell Fate and Bone-Fat Balance in Osteoporosis and Skeletal Aging by Inducing TAZ

doi: 10.1016/j.stem.2018.06.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-NFkB p65 NLS specific , Rockland , Cat#600-401-271 RRID: AB_2269511.

Techniques: Control, Recombinant, Reverse Transcription, Transfection, Red Blood Cell Lysis, Virus, Staining, Diagnostic Assay, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Software, Microscopy, Imaging

Journal: Frontiers in Nutrition

Article Title: Cannabis oil modulates liver alterations and endocannabinoid system changes in a female rat model of diet-induced MASLD

doi: 10.3389/fnut.2026.1770150

Figure Lengend Snippet: Liver NrF2 and NFκB p65 expression in female rats fed a reference diet (RD), sucrose-rich diet (SRD) or SRD with cannabis oil (SRD + Ca). (A) Representative photomicrographs of immunocytochemical staining NrF2 in the liver sections of rats. Decreased levels of nuclear and cytoplasmic positive markers are observed in the SRD group. Scale bar 50 μm. (B) Quantitative immunohistochemical analysis of liver NrF2 expression expressed as integrated optical density (IOD). (C) Representative photomicrographs of immunocytochemical staining NFκB p65 in the liver sections of rats. Increased levels of nuclear and cytoplasmic positive markers are observed in the SRD group. Scale bar 50 μm. (D) Quantitative immunohistochemical analysis of NFκB p65 expression in the liver. Data are expressed as mean ± SEM ( n = 6). Statistical differences were evaluated by one-way ANOVA followed by the Newman–Keuls post hoc test (* P < 0.05).

Article Snippet: The samples were incubated in a humid chamber first with a specific primary antibody for 4-HNE (mouse monoclonal antibody; Catalog # MAB3249; R&D Systems), NrF2 (mouse monoclonal antibody; sc-365949; Santa Cruz Biotechnology), NF-κB p65 (mouse monoclonal antibody; sc-8008; Santa Cruz Biotechnology) and TGF-β1 (mouse monoclonal antibody; sc-52893; Santa Cruz Biotechnology) (for 14–16 h at 4°C) and then with biotin-conjugated secondary antibody (anti-mouse, 1:100 dilution, Sigma) for 30 min at room temperature.

Techniques: Expressing, Cannabis, Staining, Immunohistochemical staining