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: iScience

Article Title: Cancer cell intrinsic TIM-3 induces glioblastoma progression

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet:

Article Snippet: NF-κB p65(S536) (93H1) , Cell Signaling Technology , 3033S; RRID: AB_331284.

Techniques: Control, Purification, Recombinant, Modification, Sterility, Cell Culture, Lysis, Transfection, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Imaging, Sequencing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software, Fluorescence, Microscopy

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Brazilin blocks catabolic processes in human osteoarthritic chondrocytes via inhibition of NFKB1/p50.

doi: 10.1002/jor.24013

Figure Lengend Snippet: Figure 4. Brazilin inhibits NFKB1/p50. (a) Primary human chondrocytes (n ¼ 4 donors) were left un- treated (C), treated with 10 ng/ml IL-1b for 6 h, or pre-incubated with 10 mg/ml brazilin for 1 h prior to stimulation with 10 ng/ml IL-1b for 6 h. NFKB1 mRNA levels were analyzed by RT-qPCR and expressed as relative quantities (mean SD) com- pared to controls. Significant differences compared to untreated cells (p < 0.05; n ¼ 4; Quade test). #Sig- nificant differences compared to cells treated only with IL-1b (p < 0.05; n ¼ 4; Quade test). (b) Primary human chondrocytes (n ¼ 4 donors) were left un- treated (C) or were treated with 10 ng/ml IL-1b for 15 min, 1 h, 4 h, 16 h, and 32 h. Protein extracts were subjected to Western blot analysis for the detection of p50, p65, and phosphorylated p65. b-Actin and a- tubulin were used as loading controls. The blot of one representative experiment is shown. (c) Primary chondrocytes (n ¼ 4) were left untreated (C), treated with 10 ng/ml IL-1b for 24 h, or pre-treated with 10 mg/ml brazilin for 1 h prior to stimulation with 10 ng/ml IL-1b for 24 h. Protein extracts were sub- jected to Western blot analysis for the detection of p50. b-Actin was used as loading control. The blot of one representative experiment is shown. (d) Primary chondrocytes (n ¼ 4) were left untreated (C), treated with 10 ng/ml IL-1b for 1 h, or pre-treated with 10 mg/ml brazilin for 1 h prior to stimulation with 10 ng/ml IL-1b for 1 h. Protein extracts were subjected to Western blot analysis for the detection of p65 and phosphorylated p65. a-Tubulin was used as loading control. The blot of one representative experiment is shown. (e–h) Primary chondrocytes (n ¼ 3 donors) were left untreated as controls, treated with 10 ng/ml IL-1b for 6 h, or pre-treated with 2.5, 5, and 10 mg/ml brazilin or 5, 10, and 20 mg/ml CAPE for 1 h prior to stimulation with 10 ng/ml IL-1b for 6 h. The mRNA levels of (e) IL1B, (f) TNF, (g) PTGS2, and (h) MMP13 were analyzed by RT-qPCR and expressed as percent relative to cells treated with IL-1b (100%). Significant differences compared to cells treated only with IL-1b (p < 0.05; n ¼ 3; Friedman test with many- to-one post-hoc test).

Article Snippet: After blocking, the membranes were probed for 2h with primary antibodies: Anti-NF-kB p105/p50 (1:400, ab7971, rabbit polyclonal; Abcam, Cambridge, MA), anti-NF-kB p65 (1:1,000, rabbit monoclonal; Cell Signaling Technology, Danvers, MA), anti-phospho-NF-kB p65 (Ser536, 1:1,000, rabbit monoclonal; Cell Signaling Technology), anti-a-tubulin (1:1,000, mouse monoclonal; Cell Signaling Technology) and anti-b-actin (1:5,000, mouse monoclonal; Sigma-Aldrich).

Techniques: Incubation, Quantitative RT-PCR, Western Blot, 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: International journal of molecular sciences

Article Title: WGA-M001, a Mixture of Total Extracts of Tagetes erecta and Ocimum basilicum , Synergistically Alleviates Cartilage Destruction by Inhibiting ERK and NF-κB Signaling.

doi: 10.3390/ijms242417459

Figure Lengend Snippet: Figure 4. WGA-M001 regulated OA-related molecules through dephosphorylation in the NF-κB and ERK pathways. For in silico analysis, 200 µg/mL of Tagetes erecta, Ocimum basilicum, or WGA-M001 was used for treatment of chondrocytes for 12 h along with IL-1β (1 ng/mL), and RNA sequencing was performed. (A) In each signaling pathway, the number of genes upregulated by IL-1β and then downregulated by T. erecta, O. basilicum, or WGA-M001 was shown. (B,C) Protein levels of pp38, p38, pJNK, JNK, pERK, ERK, pp65, p65, and IκB were detected by Western blot analysis and relative intensities were quantified by densitometry (n = 5). Each protein level was normalized to ERK. Data are represented as mean ± SD as results of analysis by one-way ANOVA with Dunnett’s multiple comparisons test (n = 5). * p < 0.05, **** p < 0.0001, and n.s = not significant.

Article Snippet: 2023, 24, 17459 12 of 16 cam), rabbit anti-COX-2 (ab52237; Abcam), mouse anti-IκB (9242; Cell Signaling Technology (CST), Danvers, MA, USA), mouse anti-p65 (#6956; CST), mouse anti-pp65 (#13346; CST), mouse anti-p38 (#9212; CST), mouse anti-pp38 (#9215S; CST), mouse anti-c-JNK (#9252S; CST), mouse anti-pJNK (#9251S; CST), and mouse anti-pERK (#9101S; CST).

Techniques: De-Phosphorylation Assay, In Silico, RNA Sequencing, Western Blot

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: Journal of Neurotrauma

Article Title: The Treatment of Traumatic Brain Injury with Velcade

doi: 10.1089/neu.2010.1359

Figure Lengend Snippet: The effect of Velcade on nuclear factor-κB (NF-κB) phosphorylation in the boundary zone in rats examined at 24 h after TBI. (a) Western blot detection of NF-κB and phospho-NF-κB. (b) Phospho-NF-κB (p-NF-κB) expression as detected by ELISA (n = 3, p = 0.0043; TBI, traumatic brain injury; ELISA, enzyme-linked immunosorbent assay).

Article Snippet: The PathScan Phospha-NF-κB P65 Sandwich ELISA kit (#7173; Cell Signaling Technology, Inc., Beverly, MA) was used for protein quantitative analysis on the same brain tissue used for Western blot.

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

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Anti-phospho-p65 Ser276 (Cell Signaling no. 3037) detects atypical (with MWs of 80 and 130 kDa), inducible immunoreactivities in different cell types, induced with different NF- κ B-activating agents. Cells were stimulated for increasing times (L929sA, A549, L363) or for 30 min (Raw264.7, C2C12, 1321N1) with TNF- α (2000 IU/mL), LPS (1 μ g/mL), PMA (10 ng/mL), forskolin (forsk, 10 μ M) or isoproterenol (iso, 10 μ M). For blocking experiments, lysates were loaded on one gel in duplicate and after transfer, blots were cut in half. Immunodetection was performed in parallel, using Cell Signaling no. 3037 or Cell Signaling no. 3037 preincubated for at least 30 minutes with double volume of blocking peptide (BLOCK). Arrow 1 indicates an immunoreactive band with an MW of 130 kDa; arrow 2 indicates an immunoreactive band of 80 kDa.

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Blocking Assay, Immunodetection

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: The Cell Signaling no. 3037 anti-P-p65 Ser276-immunoreactive band persists in cells where p65 is silenced via an siRNA approach. 1321N1 cells were transiently transfected with control or p65 siRNA as described in . Cells were induced for 90 min with TNF- α (2000 IU/mL), iso (10 μ M), or a combination of both stimuli. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1). 130 and 80 kDa immunoreactivities are indicated by arrows. Western Blot to detect anti-P-p65Ser536 (65 kDa arrow) was performed on the same lysates (B1). Blots A1 and B1 were extensively washed and reprobed with anti-p65 to investigate efficiency of p65 knock-down. Anti-p65 detected bands are marked with arrows (A2, B2). Finally, blots were reprobed with anti-tubulin to check loading efficiency. Anti-tubulin-detected bands are marked with arrows (A3, B3).

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Transfection, Control, Western Blot, Knockdown

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Four independent anti-P-p65 Ser276 antibodies detect inducible bands, with MWs of 80 and 130 kDa, that do not disappear upon p65 knock-down, but are inhibited when PKAc α is silenced. 1321N1 cells were transiently transfected with control, p65 or PKAc α siRNA, as described in . Cells were induced for 90 min with TNF- α (2000 IU/mL), iso (10 μ M) or a combination of both stimuli. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using SAB no. 11011 (A1), Rockland no. 100-401-264 (B1), Cell Signaling no. 3037 (B3), or a homemade anti-P-p65 Ser276 antibody (C1). 130 and 80 kDa immunoreactivities are indicated by arrows (1 and 2, resp.). Blots were extensively washed and reprobed with a mixture of anti-p65 and anti-PKAc α to investigate knock-down efficiency. Anti-p65-detected bands are marked by the upper arrow, anti-PKAc α -reactive bands are marked by the lower arrow (A2, B2, C2). Tubulin loading controls are added as Supplementary Figure 1.

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Knockdown, Transfection, Control, Western Blot

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: Presence of anti-P-p65 Ser276-reactive bands in p65 deficient MEF cells. p65 knock-out MEF cells (−/−), or p65 −/− MEF cells reconstituted with wild type p65 (p65 wt) or p65 with Ser276 mutated to alanine (p65 S/A), were induced with forskolin (forsk, 10 μ M) for 0, 30, and 60 min. Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1) or SAB no. 11011 (B1). Blots were extensively washed and reprobed with anti-p65 to confirm p65 absence in −/− MEFs, and p65 presence in wt and S/A reconstituted MEFs (A2 and B2, arrows indicate anti-p65 detected bands).

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Knock-Out, Western Blot

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: The detected 130 kDa and 80 kDa immunoreactivities are not NF- κ B p105/p50 or c-Rel. 1321N1 cells were transiently transfected with control, p105/p50 or c-Rel siRNA as described in . Cells were induced for 30 min with TNF- α (2000 IU/mL) or iso (10 μ M). Presence of Ser276-phosphorylated p65 in cell lysates was analysed by Western Blot using Cell Signaling no. 3037 (A1, B1). 130 and 80 kDa immunoreactivities are indicated by arrows (1 and 2 resp.). Because of the overlap of the 130 kDa and the p105 immunoreactive bands, blot A was first stripped and reprobed with anti-p105/p50 (A2). p105 immunoreactivity is indicated by the upper arrow; p50 immunoreactivity is indicated by the lower arrow. Blot B was extensively washed and reprobed (without stripping) with anti-c-Rel (B2). c-Rel immunoreactivity is indicated by an arrow. Tubulin was used as a loading control (A3, B3).

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: Transfection, Control, Western Blot, Stripping Membranes

Journal: Journal of Biomedicine and Biotechnology

Article Title: Hunting for Serine 276-Phosphorylated p65

doi: 10.1155/2010/275892

Figure Lengend Snippet: In vitro recognition of the phosphorylated Serine 276 residue of p65 by Cell Signaling no. 3037. Recombinant wt p65-GST or SC mutant p65-GST fusion proteins were in vitro phosphorylated using recombinant MSK-1. 250 ng of the recombinant proteins were spotted on a nitrocellulose membrane and subjected to Western Blotting with Ser276 phospho-specific p65 antibody, either preincubated with 10 μ g/mL phosphorylated peptide that was used to generate the antibody (upper panel) or not. 1 μ g of recombinant protein was separated by SDS-PAGE after the in vitro kinase assays performed either in the presence of active MSK-1 and/or 10 μ M H89 (lower panel) or not.

Article Snippet: Four rabbit polyclonal antibodies directed against the phosphorylated Ser276 residue of p65 were used in this study: anti-P-p65 Ser276 (no. 3037) from Cell Signaling (Danvers, MA), anti-P-p65 Ser276 (no. 11011) from Signalway Antibody (SAB, Pearland, TX), anti-P-p65 Ser276 (no. 100-401-264) from Rockland (Gilbertsville, PA), and a homemade anti-P-p65 Ser276 antibody (kind gift of Prof. Dr. P. Cohen, University of Dundee, UK; produced and purified by Drs. C. Armstrong and J. Leitch).

Techniques: In Vitro, Residue, Recombinant, Mutagenesis, Membrane, Western Blot, SDS Page