Journal: International Journal of Dentistry

Article Title: Acinar Cell Proliferation Promoted by BMP2 in Injured Mouse Parotid Gland: BMP2 Promotes Cell Proliferation in Parotid Gland

doi: 10.1155/2023/1765317

Figure Lengend Snippet: Mouse parotid acinar cells in primary culture. (a) Cell proliferative capacity of BMP2-added (100 ng/mL) and nonadded (control) groups after 48 hr of culture. The proliferative capacity of BMP2-added cultured cells was significantly increased compared to that in the control group. ∗∗ Indicates significance at P < 0.01, ∗ P < 0.05. Data were shown as mean ± SD. Three independent experiments were performed. (b) Protein expression of E-cadherin (an epithelial marker) and vimentin (a mesenchymal marker) in cultured cells of each group at 48 hr after the addition of BMP2. NIH3T3 (3T3) was used as a positive control for mesenchymal markers. RS: rat serum.

Article Snippet: NIH3T3 whole cell lysate (Novus Biologicals, Centennial, CO, USA) was used as a positive control for mesenchymal markers.

Techniques: Control, Cell Culture, Expressing, Marker, Positive Control

Journal: STAR Protocols

Article Title: An optimized chromatin immunoprecipitation protocol using Staph-seq for analyzing genome-wide protein-DNA interactions

doi: 10.1016/j.xpro.2022.101918

Figure Lengend Snippet:

Article Snippet: V6.5 mouse embryonic stem cells , Novus Biologicals , Cat#NBP1-41162.

Techniques: Recombinant, SYBR Green Assay, Next-Generation Sequencing, Agarose Gel Electrophoresis, Purification, Software, Real-time Polymerase Chain Reaction

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Expression and coding potential analysis of Hmrhl. a. Quantitative real time PCR analysis of Hmrhl expression showed that it is expressed in all human tissues (Brain, Heart, Kidney, lung, liver, pancreas, spleen, thymus, small intestine, colon, skeletal muscle, testes, prostate, ovary, placenta, leukocyte, from left to right) examined. Lowest expression was found in skeletal muscle (SM) which was taken as control, the level of which was considered as 1 and all others were plotted in comparison to it. Highest expression was seen in spleen (spln) followed by pancreas (Pnc), testis (Tst) and other tissues. b. Northern blot detection of Hmrhl. Total RNA from HEK 293T and K562 cell lines were separated on agarose gel and subsequently hybridized with DIG labelled Hmrhl specific riboprobe to detect the transcript (i). In parallel, methylene blue staining was used to determine the size of HMRHL, using 28 S rRNA (5 kb) and 18s rRNA (1.9 kb) as reference (ii). Note that the size of Hmrhl is similar to that of 28s rRNA, revealing that Hmrhl is about 5 kb in size. c. Protein-coding potential as determined by Broad Institute's PhyloCSF data and visualized in UCSC Genome Browser, showing that Hmrhl has no coding potential. d. Circular phylogenetic tree built in iTOL (Interactive Tree of Life).

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Comparison, Northern Blot, Agarose Gel Electrophoresis, Staining

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl locus exhibits hallmarks of enhancer. a. ENCODE data visualized through Integrated Genome Viewer (IGV) for DNase hypersensitive sites, p300 binding, enhancer specific histone marks, H3K27Ac and H3K4Me1 and the promoter specific histone mark, H3K4Me3 at the 5′ end of Hmrhl, only in K562 but not in GM12878 cells. Note the two prominent peaks (red) for the enhancer mark H3K27Ac in K562. b-c. Chromatin immunoprecipitation with Ab8895 (anti-H3K4Me1 antibody) and Ab4729 (anti-H3K27Ac antibody) followed by qPCR in K562 cells. Note the enrichment of both the enhancer marks at the 5′ end of Hmrhl in the IP fraction as compared to input/PIS/gene desert region (GD), that serves as a negative control.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Negative Control

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl locus exhibits hallmarks of enhancer contd. a. Encode data shows the binding of various transcription and PolII at the 5′ end of Hmrhl. We have retained the H3K27Ac peaks in this figure also for a reference. b. Schematic for chromatin interaction analysis (ChiaPET data) for Hmrhl. The large purple-black peak representing histone marks on the extreme left denotes the promoter of phkb gene while the small purple peak at the far right represents the 5'end of Hmrhl. ChiaPET data shows the interaction of Hmrhl locus with phkb promoter, as represented by two black boxes (blue arrows) connected by a black line in b. The Hmrhl locus is expanded below in c , showing that this locus has enhancer properties only in K562 cell line (orange-yellow color), but not in other cell lines like GM12878, HepG2 or hESC. Genomic segments are colour coded by ENCODE as denoted in d , with red colour signifying active promoter ( phkb promoter at far left, black arrow in b ) while orange colour represents active enhancer at Hmrhl locus at far right (red arrow in b ).

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Binding Assay

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl is differentially expressed in various cancers. a. Expression of Hmrhl in various normal and cancer samples as observed by qPCR. Note that Hmrhl is highly upregulated in several lymphoma samples (bracket) in comparison to normal range (arrow). In fact, of all cancers, the highest levels of Hmrhl are seen in some of the lymphoma samples. b-c. qPCR analysis of Hmrhl and PHKB expression showing that both are over expressed in K562 leukemia condition as compared to GM12878 normal lymphocytes.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Expressing, Comparison

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl functions as enhancer RNA for phkb gene. a. Lucifaerase assay showing the intense signal of reporter activity in K562 cells with insert 3 cloned in enhancer vector. Note the low level of luciferase signal obtained with insert 2 both with promoter and enhancer vectors. b. siRNA (Sigma) mediated down-regulation of Hmrhl causes down-regulation of PHKB in K562 cells treated with Hmrhl specific siRNA pool as compared to control cells without transfection and cells treated with scrambled siRNA as negative control. c-d. Smart pool siRNA (Dharmacon) were used against the Hmrhl region to downregulate Hmrhl and subsequently expression level of PHKB gene were checked by qPCR in both K562 and GM12878 cell lines. Scrambled siRNA was used as a negative control. Note the down regulation of PHKB only in K562.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Activity Assay, Clone Assay, Plasmid Preparation, Luciferase, Control, Transfection, Negative Control, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Antibody validation for Western blot: By the user, for the user

doi: 10.1074/jbc.RA119.010472

Figure Lengend Snippet: Validation of IDH1 antibody using purified recombinant protein in multicolor and chemiluminescent Western blotting. Multicolor and chemiluminescent Western blottings were performed using 10% Bis-Tris SDS-polyacrylamide gel and MOPS buffer system to validate the IDH1 antibody using a purified recombinant IDH1 protein (0.16 μg) containing a c-Myc tag in addition to HEK293T and HeLa whole-cell lysates. A, c-Myc protein tag present on the purified IDH1 recombinant protein is detected in the 700-nm channel ( red ) at 50 kDa via mouse anti-c-Myc antibody (ab32;1 μg/ml) using IRDye 680RD goat anti-mouse IgG (H + L) for detection. Some overspill of the recombinant protein into neighboring lanes is observed ( white box ). B, IDH1 recombinant protein and endogenous IDH1 protein, present in HEK293T and HeLa, is detected in the 800-nm channel ( green ) at 55 and 50 kDa, respectively, using rabbit anti-IDH1 antibody (ab172964; 1.2 μg/ml) and IRDye 800CW goat anti-mouse IgG (H + L) for detection. C, when both 700- and 800-nm channels are displayed, the signal from ab32 and ab172964 overlaps at 50 kDa, identifying the c-Myc–tagged IDH1 protein. No overlap is seen for the endogenous IDH1 present in HEK293T and HeLa whole-cell lysates. A–C , lysates loaded per lane are as follows: 20 μg of blocking buffer: Odyssey blocking buffer (TBS); imager: Odyssey® CLx; resolution: 169 μm; intensity: auto mode. Chameleon TM Duo pre-stained protein ladder for accurate sizing of protein bands. D, single blot was split into two halves ( green line ) to be incubated with either rabbit anti-IDH1 antibody (ab172964; 0.115 μg/ml) or the corresponding rabbit monoclonal IgG isotype control (ab172730; 0.166 μg/ml) to detect the endogenous IDH1 protein present in HeLa and HEK293T as well IDH1 recombinant protein. Both halves were incubated with HRP-conjugated goat anti-mouse IgG (H + L). E, single blot was split into two halves ( green line ) to be incubated with either mouse anti-c-Myc antibody (ab32; 1 μg/ml) or the corresponding mouse monoclonal IgG1 isotype control (ab18443; 1 μg/ml) to detect c-Myc protein tag present on the purified IDH1 recombinant protein but absent in HEK293T and HeLa whole-cell lysates. Both halves were incubated with HRP-conjugated goat anti-rabbit IgG (H + L). Blots were detected with WesternSure® PREMIUM chemiluminescent substrate (LI-COR 926–95000) and imaged on an Odyssey® Fc with the following resolution: 125 μm and exposure of 2 min. Lysate loaded per lane: 20 μg; protein ladder: WesternSure® pre-stained chemiluminescent protein ladder (LI-COR 926-980000); blocking buffer: intercept blocking buffer (TBS); intercept T20 (TBS) antibody diluent.

Article Snippet: Isocitrate dehydrogenase (IDH1) (NM_005896) human recombinant protein (OriGene no. TP310582), IDH1 (NM_005896) human overexpression lysate supplied with parental HEK293T lysate (OriGene no. LY401782; HEK293T LY500001; lot no. 0076CF), and IDH1 knockout cell lysate (supplied with parental HeLa control lysate) (Origene no. LC810112, LC810Hela; lot no. 1601) were mixed with either 2× protein loading buffer (PLB) (LICOR no. 928-40004) or 2× SDS buffer (OriGene) and denatured by boiling at 97 °C for 5 min.

Techniques: Biomarker Discovery, Purification, Recombinant, Western Blot, Blocking Assay, Staining, Incubation, Control

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 1. TAK1 regulates Nod2-mediated NF-B activation. A, the dominant negative form of TAK1 (TAK1DN) inhibits NF-B activation induced by Nod2. HEK293T cells were transfected with pCDNA-Nod2 (3 ng) and reporter constructs RSV-KB-Luc and RSV--galactosidase plus the dominant negative forms of TBK1, Ubc13, Rip, or TAK1 (120 ng each) or control vector. Luciferase activity was determined 24 h after transfection and normalized on the basis of -galactosidase activity. All experiments were performed in triplicate and repeated at least three times with equivalent results. *, p 0.0001 (vector versus TAK1DN). B, the dose effect of TAK1DN on Nod2-mediated NF-B. HEK293T cells were transfected with fixed amounts of pCDNA-Nod2 (3 ng), RSV-KB- Luc, and RSV--galactosidase plus the indicated doses of TAK1DN. The luciferase activity of cells transfected with reporters and Nod2 alone (control) was arbitrarily set at 100, and the percent of inhibition was calculated accordingly. The experiments were performed in triplicate and repeated at least three times with equivalent results.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Activation Assay, Dominant Negative Mutation, Transfection, Construct, Control, Plasmid Preparation, Luciferase, Activity Assay, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 2. Dominant negative form of TAK1 (TAK1DN) inhibits MDP-induced NF-B activation in Nod2-expressing cells. HEK293T cells were transfected with pCDNA-Nod2 (5 ng) and reporter constructs plus the indicated amounts of TAK1DN or control vector. Cells were stimulated with MDP (100 ng/ml) 8 h after transfection. Luciferase activity was determined 16 h after stimulation and normal- ized on the basis of -galactosidase activity. All experiments were performed in triplicate and repeated three times with equivalent re- sults. *, p 0.05.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Dominant Negative Mutation, Activation Assay, Expressing, Transfection, Construct, Control, Plasmid Preparation, Luciferase, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 3. Nod2 interacts with TAK1. A, Nod2 coimmunoprecipitates with TAK1. HEK293T cells were cotransfected with plasmid pCMV-HA- TAK1 (4 g) plus 4 g of DNA pLPCX-FLAG-Nod2, control vector pLPCX-FLAG, or pLPCX-FLAG-LRR. Immunoprecipitations (IP) were performed with anti-HA antibodies and subjected to Western blot (IB) analysis using anti-FLAG antibodies to detect FLAG-Nod2 or FLAG-LRR (left). Immunoprecipitations were also performed using anti-FLAG antibodies and subjected to Western blot analysis using anti-HA antibodies to detect TAK1 (right). All experiments were repeated three times with equivalent results. B, coprecipitation of endogenous TAK1 and Nod2. LS174T and THP-1 cell lysates were prepared as described under “Experimental Procedures.” Immunoprecipitations were performed with anti-Nod2 antibodies or control Ig and subjected to Western blot analysis using anti-TAK1 antibodies (top panel). The membranes were reprobed with anti-Nod2 (middle panel). Aliquots of cell lysates were immunoblotted with anti-TAK1 (bottom panel). C, the LRR region of Nod2 interacts with TAK1. Full-length TAK1 translated in vitro and labeled with 35S was incubated with GSTLRR fusion protein, GSTCARD, or GST protein. Input TAK1 indicates 1/10 of 35S-labeled protein used in each GST pull-down assay. All experiments were repeated two times with equivalent results. D, the N-terminal of TAK1 interacts with the LRR region of Nod2. Full-length (FL) and truncated forms of TAK1 (aa 1–303, 1–403, and 286–632) were generated by in vitro transcription and translation and incubated with GSTLRR or GST protein. Input TAK1 indicates 1/10 of 35S-labeled protein used in each GST pull-down assay. All experiments were repeated three times with equivalent results.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Plasmid Preparation, Control, Western Blot, In Vitro, Labeling, Incubation, Pull Down Assay, Generated

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 5. Nod2 does not induce TAK1 activation. HEK293T cells were transfected with equal amounts of the indicated expression con- structs: control vector and TAK1 (leftmost lane), Nod2 and HA-TAK1 (3 g) (middle lane), TAB1 and TAK1 (rightmost lane). TAK1 protein was immunoprecipitated with anti-TAK1 antibody and subjected to phos- phorylation as described under “Experimental Procedures.” All experi- ments were repeated two times with equivalent results.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Activation Assay, Transfection, Expressing, Control, Plasmid Preparation, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 6. Coexpression of Nod2 and TAK1 does not exert a syn- ergistic effect on NF-B activation. HEK293T cells were trans- fected with Nod2 (5 ng) alone, TAK1 (25 ng), and TAB1 (2 ng) together, TAK1 (25 ng) and TAB1 (2 ng) plus Nod2 (5 ng), or TAK1 (25 ng) and TAB1 (2 ng) plus control vector (5 ng). Expression plasmids RSV-KB- Luc and RSV--galactosidase were included in each transfection. Lu- ciferase activity was determined 24 h after transfection and normalized on the basis of -galactosidase activity. All experiments were performed in triplicate and were repeated three times with equivalent results.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Activation Assay, Control, Plasmid Preparation, Expressing, Transfection, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 7. Nod2 suppresses TAK1-induced NF-B activation. A, TAK1 induces NF-B activation in RICK/ fibroblasts. RICK/ fibroblasts were seeded in 12-well plates (105/well). Reporter constructs with TAK1 (25 ng) and TAB1 (2 ng) and without (none) were cotransfected into cells by FuGENE 6. B, TAK1 activates NF-B equally well in both RICK/ and wild-type fibroblasts. RICK wild type (wild, shaded bar) and Rick/ (null, solid bar) fibroblasts were transfected with the indicated amounts of TAK1 and TAB1, along with the reporter constructs. Luciferase activity was determined 24 h after transfection and normalized on the basis of -galactosidase activity. All experiments were performed in triplicate and repeated two times with equivalent results. C, Nod2 inhibits TAK1-induced NF-B activation in RICK/ fibroblasts. RICK/ fibroblasts were transfected with Nod2 alone (LPCX-Nod2, 50 ng) or with TAK1 (25 ng) and TAB1 (2 ng) plus the indicated amounts of Nod2 (LPCX-Nod2, 0, 25, 50 ng) or control vector (LPCX, 25, 50 ng). Luciferase activity was determined 24 h after transfection and normalized on the basis of -galactosidase activity. All experiments were performed in triplicate and repeated three times with equivalent results. *, p 0.01 (Nod2 versus control vector). D, Nod2 siRNA efficiently blocks expression of transfected Nod2. HEK293T cells were transiently transfected with Nod2-specific siRNA or control siRNA, and 24 h later, they were transfected with FLAG-Nod2. Cell lysates were prepared 24 h after the Nod2 transfection and subjected to SDS-PAGE and immunoblotting by anti-FLAG antibody. The arrows indicate the Nod2 and nonspecific (NS) bands. E, expression of Nod2 siRNA suppresses endogenous NF-B activity. LS174T cells were transfected with either Nod2-specific siRNA or control siRNA plus the reporter constructs. Luciferase activity was determined 24 h after transfection as in C. All experiments were performed in triplicate and repeated three times with equivalent results. *, p 0.05. F, expression of Nod2 siRNA potentiates TAK1-induced NF-B activation. LS174T cells were transfected with Nod2-specific siRNA or control siRNA. These cells were then transfected with TAK1 (25 ng), TAB1 (2 ng), and the reporter constructs 24 h after the siRNA transfection. Luciferase activity was analyzed as in C. The results represent the mean of two independent experiments. Increase in NF-B activation in the control siRNA group was arbitrarily set at 1.

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Activation Assay, Construct, Transfection, Luciferase, Activity Assay, Control, Plasmid Preparation, Expressing, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Cross-talk between Nod2 and TAK1 Signaling Pathways

doi: 10.1074/jbc.m400682200

Figure Lengend Snippet: FIG. 8. Inhibitory effect of the LRR region on TAK1-induced NF-B acti- vation. A, the LRR region of Nod2 sup- presses TAK1-induced NF-B activation. HEK293T cells were transfected with TAK1 (25 ng) and TAB1 (2 ng) plus LRR in the amounts indicated. HEK293T cells transfected with identical amounts of TAK1 and TAB1 and control vector were used as the control. Luciferase activity was determined 24 h after transfection and normalized on the basis of -galacto- sidase activity. All experiments were per- formed in triplicate and repeated three times with equivalent results. B, mutant LRR is less effective than wild-type LRR at suppressing TAK1-mediated NF-B ac- tivation. HEK293T cells were transfected with TAK1 (25 ng) and TAB1 (2 ng) plus wild-type or mutant LRR in the amounts indicated (left). Luciferase activity was determined 24 h after transfection and normalized on the basis of -galactosidase activity. All experiments were performed in triplicate and repeated three times with equivalent results. *, p 0.05 (LRR versus mutant LRR). A cell lysate fraction used for the luciferase assay was sub- jected to Western blot analysis to deter- mine the expression of TAK1, TAB1, and LRR, both wild type and mutant (right).

Article Snippet: Lysates of the HEK293 cells that had been transfected with various expression constructs and HA-TAK1 were immunoprecipitated with anti-TAK1 antibody (Santa Cruz Biotechnology) for 1 h. Immunoprecipitates were incubated for 2 min at 25 °C in 10 l of kinase buffer containing 10 mM Hepes (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 5 Ci of [ - 32P]ATP (3000 Ci/mmol).

Techniques: Activation Assay, Transfection, Control, Plasmid Preparation, Luciferase, Activity Assay, Mutagenesis, Western Blot, Expressing