tab1  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: Src directly phosphorylates TAB1-Y481. A , the domain structure of TAB1 and the amino acid sequence around Y481. Conserved phosphorylation serine/threonine and tyrosine sites in the C-terminal regions are highlighted in green / blue and red . B , COS-7 cells were transfected with EGFP-tagged full-length (FL), N-terminal (N), or C-terminal (C) TAB1 and Src. C , COS-7 cells were transfected with EGFP-TAB1 and Src. Dasatinib (0.1 μM) was added 1 h before cells were harvested. D , COS-7 cells were transfected with TAB1-C or its Y481F mutant (YF) and Src. Cell lysates and immunoprecipitates with an anti-GFP antibody were immunoblotted with anti-phosphotyrosine and anti-GFP antibodies. The expression of active Src was detected by an anti-phospho-Src family kinase (Y419) antibody ( B – D ). E , an in vitro kinase assay was performed using recombinant GST-TAB1-C and active GST-Src proteins. F , COS-7 cells were transfected with EGFP-TAB1 with SFKs, including Src, Fyn, Lyn, and Lck. G , HCT-116-Src cells were treated with 10 ng/ml doxycycline (Dox) for 24 h. TAB1-Y481 phosphorylation and other proteins were detected by an anti-phospho-Y481-TAB1 antibody and the antibodies described above, respectively ( E – G ). H , the relative quantification of pY481-TAB1, normalized to total TAB1, is presented as the mean ± SD from three independent experiments. p values were calculated using Welch’s two-tailed t test. ∗ p < 0.05.

Article Snippet: The synthetic pY481-TAB1 peptide (pY-TAB1; EDGRVEP[pY]VDFAEFY), biotinylated pY-TAB1 peptide, and KLH-conjugated pY-TAB1 peptide were obtained from Eurofins (Tokyo, Japan).

Techniques: Sequencing, Phospho-proteomics, Transfection, Mutagenesis, Expressing, In Vitro, Kinase Assay, Recombinant, Quantitative Proteomics, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: Src phosphorylates free TAB1 not bound to TAK1. A , Crystal structure of the TAK1-TAB1 fusion protein (PDB id: 2EVA ). An enlarged version of the interaction interface shows Y125 and R225 of the TAK1 kinase domain forming a hydrogen bond ( yellow dashed line ) with TAB1-Y481. The distance between the backbone amide nitrogen atom of Y125 and the side chain oxygen atom of Y481 is 3.4 Å, while the distance between the guanidinium nitrogen atom (Nη1) of R225 and the side chain oxygen atom of Y481 is 3.1 Å. The figure was generated using PyMol (DeLano Scientific; www.pymol.org ). B , COS-7 cells were transfected with EGFP-TAB1-C or TAK1-TAB1 with Src. C , COS-7 cells were transfected with EGFP-TAB1, Flag-TAK1, and Src. Cell lysates or anti-GFP immunoprecipitates were immunoblotted with primary antibodies against pY481-TAB1, EGFP, pT187-TAK1, TAK1, and pY419-Src. D , the relative quantification of pY481-TAB1 in lysates, normalized to total TAB1, is presented as the mean ± SD of three independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗∗ p < 0.01.

Article Snippet: The synthetic pY481-TAB1 peptide (pY-TAB1; EDGRVEP[pY]VDFAEFY), biotinylated pY-TAB1 peptide, and KLH-conjugated pY-TAB1 peptide were obtained from Eurofins (Tokyo, Japan).

Techniques: Generated, Transfection, Quantitative Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: p38 enhances Src-mediated TAB1-Y481 phosphorylation. A and C , COS-7 cells were transfected with EGFP-TAB1, Src, and the wild-type (WT) or kinase-dead (KD) mutant of Flag-p38α. SB203580 (10 μM) was added for 5 h ( C ). B , The relative quantification of pY481-TAB1, normalized to total TAB1, is presented as the mean ± SD of three independent experiments. D – F , COS-7 cells transfected with EGFP-TAB1 (WT or YF) were stimulated with 1 mM H 2 O 2 for the indicated time. SB203580 was added as a pre-treatment for 5 h. G , COS-7 cells were pretreated with baricitinib (0.5 μM), SB203580 (10 μM), and saracatinib (0.5 μM) for 1 h and then treated with 1 mM H 2 O 2 for 5 min. H , The relative quantification of pY701-STAT1, normalized to total-STAT1, is presented as the mean ± SD of three independent experiments. Cell lysates were immunoblotted with primary antibodies indicated ( A and C – G ). p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The synthetic pY481-TAB1 peptide (pY-TAB1; EDGRVEP[pY]VDFAEFY), biotinylated pY-TAB1 peptide, and KLH-conjugated pY-TAB1 peptide were obtained from Eurofins (Tokyo, Japan).

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Quantitative Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: The p38-mediated enhancement of pY481-TAB1 is independent of the serine/threonine phosphorylation of TAB1 by p38. A – C , COS-7 cells were transfected with Myc-tagged TAB1 (STS/AAA and 4S/A in a) or EGFP-tagged TAB1 (WT or CS/NCS in B ), Src, and p38α. D , COS-7 cells were transfected with HA-tagged FAK, Src, and p38α. Cell lysates were immunoblotted with the primary antibodies indicated. E , the relative quantification of pY576/577-FAK, normalized to total FAK, is presented as the mean ± SD of three independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗∗ p < 0.01.

Article Snippet: The synthetic pY481-TAB1 peptide (pY-TAB1; EDGRVEP[pY]VDFAEFY), biotinylated pY-TAB1 peptide, and KLH-conjugated pY-TAB1 peptide were obtained from Eurofins (Tokyo, Japan).

Techniques: Phospho-proteomics, Transfection, Quantitative Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: p38 directly phosphorylates Src to increase its kinase activity. A , comparison between the substrate consensus sequence of p38α and the amino acid sequences of human Src around S75. Asterisks indicate amino acids that are common to both. B , an in vitro kinase assay using recombinant His-tagged Src and GST-tagged p38α proteins. Immunoblot analyses were performed with the primary antibodies indicated. C , the relative quantification of pS75-Src and pY419-Src, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by Welch’s two-tailed t test was applied. ∗∗ p < 0.01. D , COS-7 cells were transfected with Src and p38α (WT, CA or KD), and then treated with SB203580 for 5 h. Immunoblot analyses were performed with the primary antibodies indicated. E , the relative quantification of pS75-Src and pY419-Src, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗∗ p < 0.01. F , Src immunoprecipitated from transfected COS-7 cells was preincubated with recombinant p38 for 60 min. After removal of p38, the tyrosine kinase activity of Src toward GST-TAB1-C at Y481 was evaluated by an additional 20-min incubation. The relative quantification of pY481-TAB1, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by Welch’s two-tailed t test was applied. ∗ p < 0.05. G , COS-7 cells were transfected with EGFP-TAB1, Src (WT or S75A), and p38α. Immunoblot analyses were performed with the primary antibodies indicated. H , the relative quantification of pY481-TAB1, normalized to total TAB1, is presented as the mean ± SD of four independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗ p < 0.05.

Article Snippet: The synthetic pY481-TAB1 peptide (pY-TAB1; EDGRVEP[pY]VDFAEFY), biotinylated pY-TAB1 peptide, and KLH-conjugated pY-TAB1 peptide were obtained from Eurofins (Tokyo, Japan).

Techniques: Activity Assay, Comparison, Sequencing, In Vitro, Kinase Assay, Recombinant, Western Blot, Quantitative Proteomics, Two Tailed Test, Transfection, Immunoprecipitation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: Differences in TAB1 and FAK recognition patterns by Src . A – C , COS-7 cells were transfected with EGFP-tagged TAB1 and Src (WT, Y419F and R178A) and active p38α-CA. D – F , COS-7 cells were transfected with HA-tagged FAK and Src (WT, Y419F and R178A) and active p38α-CA. Cell lysates were immunoblotted with primary antibodies as indicated. The relative quantification of pY481-TAB1/TAB1 and pY576/577-FAK/FAK, respectively, is presented as the mean ± SD of three independent experiments. p values were calculated by Dunnett’s multiple comparisons test was applied. ∗∗ p < 0.01.

Article Snippet: The synthetic pY481-TAB1 peptide (pY-TAB1; EDGRVEP[pY]VDFAEFY), biotinylated pY-TAB1 peptide, and KLH-conjugated pY-TAB1 peptide were obtained from Eurofins (Tokyo, Japan).

Techniques: Transfection, Quantitative Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: Src directly phosphorylates TAB1-Y481. A , the domain structure of TAB1 and the amino acid sequence around Y481. Conserved phosphorylation serine/threonine and tyrosine sites in the C-terminal regions are highlighted in green / blue and red . B , COS-7 cells were transfected with EGFP-tagged full-length (FL), N-terminal (N), or C-terminal (C) TAB1 and Src. C , COS-7 cells were transfected with EGFP-TAB1 and Src. Dasatinib (0.1 μM) was added 1 h before cells were harvested. D , COS-7 cells were transfected with TAB1-C or its Y481F mutant (YF) and Src. Cell lysates and immunoprecipitates with an anti-GFP antibody were immunoblotted with anti-phosphotyrosine and anti-GFP antibodies. The expression of active Src was detected by an anti-phospho-Src family kinase (Y419) antibody ( B – D ). E , an in vitro kinase assay was performed using recombinant GST-TAB1-C and active GST-Src proteins. F , COS-7 cells were transfected with EGFP-TAB1 with SFKs, including Src, Fyn, Lyn, and Lck. G , HCT-116-Src cells were treated with 10 ng/ml doxycycline (Dox) for 24 h. TAB1-Y481 phosphorylation and other proteins were detected by an anti-phospho-Y481-TAB1 antibody and the antibodies described above, respectively ( E – G ). H , the relative quantification of pY481-TAB1, normalized to total TAB1, is presented as the mean ± SD from three independent experiments. p values were calculated using Welch’s two-tailed t test. ∗ p < 0.05.

Article Snippet: Primary antibodies for p38α (sc-271120), TAB1 (sc-166138), phosphotyrosine (clone PY20, sc-508), GFP (sc-9996), and β-actin (sc-47778) were obtained from Santa Cruz Biotechnology.

Techniques: Sequencing, Phospho-proteomics, Transfection, Mutagenesis, Expressing, In Vitro, Kinase Assay, Recombinant, Quantitative Proteomics, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: Src phosphorylates free TAB1 not bound to TAK1. A , Crystal structure of the TAK1-TAB1 fusion protein (PDB id: 2EVA ). An enlarged version of the interaction interface shows Y125 and R225 of the TAK1 kinase domain forming a hydrogen bond ( yellow dashed line ) with TAB1-Y481. The distance between the backbone amide nitrogen atom of Y125 and the side chain oxygen atom of Y481 is 3.4 Å, while the distance between the guanidinium nitrogen atom (Nη1) of R225 and the side chain oxygen atom of Y481 is 3.1 Å. The figure was generated using PyMol (DeLano Scientific; www.pymol.org ). B , COS-7 cells were transfected with EGFP-TAB1-C or TAK1-TAB1 with Src. C , COS-7 cells were transfected with EGFP-TAB1, Flag-TAK1, and Src. Cell lysates or anti-GFP immunoprecipitates were immunoblotted with primary antibodies against pY481-TAB1, EGFP, pT187-TAK1, TAK1, and pY419-Src. D , the relative quantification of pY481-TAB1 in lysates, normalized to total TAB1, is presented as the mean ± SD of three independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗∗ p < 0.01.

Article Snippet: Primary antibodies for p38α (sc-271120), TAB1 (sc-166138), phosphotyrosine (clone PY20, sc-508), GFP (sc-9996), and β-actin (sc-47778) were obtained from Santa Cruz Biotechnology.

Techniques: Generated, Transfection, Quantitative Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: p38 enhances Src-mediated TAB1-Y481 phosphorylation. A and C , COS-7 cells were transfected with EGFP-TAB1, Src, and the wild-type (WT) or kinase-dead (KD) mutant of Flag-p38α. SB203580 (10 μM) was added for 5 h ( C ). B , The relative quantification of pY481-TAB1, normalized to total TAB1, is presented as the mean ± SD of three independent experiments. D – F , COS-7 cells transfected with EGFP-TAB1 (WT or YF) were stimulated with 1 mM H 2 O 2 for the indicated time. SB203580 was added as a pre-treatment for 5 h. G , COS-7 cells were pretreated with baricitinib (0.5 μM), SB203580 (10 μM), and saracatinib (0.5 μM) for 1 h and then treated with 1 mM H 2 O 2 for 5 min. H , The relative quantification of pY701-STAT1, normalized to total-STAT1, is presented as the mean ± SD of three independent experiments. Cell lysates were immunoblotted with primary antibodies indicated ( A and C – G ). p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Primary antibodies for p38α (sc-271120), TAB1 (sc-166138), phosphotyrosine (clone PY20, sc-508), GFP (sc-9996), and β-actin (sc-47778) were obtained from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Quantitative Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: The p38-mediated enhancement of pY481-TAB1 is independent of the serine/threonine phosphorylation of TAB1 by p38. A – C , COS-7 cells were transfected with Myc-tagged TAB1 (STS/AAA and 4S/A in a) or EGFP-tagged TAB1 (WT or CS/NCS in B ), Src, and p38α. D , COS-7 cells were transfected with HA-tagged FAK, Src, and p38α. Cell lysates were immunoblotted with the primary antibodies indicated. E , the relative quantification of pY576/577-FAK, normalized to total FAK, is presented as the mean ± SD of three independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗∗ p < 0.01.

Article Snippet: Primary antibodies for p38α (sc-271120), TAB1 (sc-166138), phosphotyrosine (clone PY20, sc-508), GFP (sc-9996), and β-actin (sc-47778) were obtained from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Transfection, Quantitative Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: p38 directly phosphorylates Src to increase its kinase activity. A , comparison between the substrate consensus sequence of p38α and the amino acid sequences of human Src around S75. Asterisks indicate amino acids that are common to both. B , an in vitro kinase assay using recombinant His-tagged Src and GST-tagged p38α proteins. Immunoblot analyses were performed with the primary antibodies indicated. C , the relative quantification of pS75-Src and pY419-Src, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by Welch’s two-tailed t test was applied. ∗∗ p < 0.01. D , COS-7 cells were transfected with Src and p38α (WT, CA or KD), and then treated with SB203580 for 5 h. Immunoblot analyses were performed with the primary antibodies indicated. E , the relative quantification of pS75-Src and pY419-Src, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗∗ p < 0.01. F , Src immunoprecipitated from transfected COS-7 cells was preincubated with recombinant p38 for 60 min. After removal of p38, the tyrosine kinase activity of Src toward GST-TAB1-C at Y481 was evaluated by an additional 20-min incubation. The relative quantification of pY481-TAB1, normalized to total Src, is presented as the mean ± SD of three independent experiments. p values were calculated by Welch’s two-tailed t test was applied. ∗ p < 0.05. G , COS-7 cells were transfected with EGFP-TAB1, Src (WT or S75A), and p38α. Immunoblot analyses were performed with the primary antibodies indicated. H , the relative quantification of pY481-TAB1, normalized to total TAB1, is presented as the mean ± SD of four independent experiments. p values were calculated by one-way ANOVA followed by either Tukey’s HSD test was applied. ∗ p < 0.05.

Article Snippet: Primary antibodies for p38α (sc-271120), TAB1 (sc-166138), phosphotyrosine (clone PY20, sc-508), GFP (sc-9996), and β-actin (sc-47778) were obtained from Santa Cruz Biotechnology.

Techniques: Activity Assay, Comparison, Sequencing, In Vitro, Kinase Assay, Recombinant, Western Blot, Quantitative Proteomics, Two Tailed Test, Transfection, Immunoprecipitation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The stress-activated kinase p38 mediates non-canonical activation of Src and tyrosine phosphorylation of the adapter protein TAB1

doi: 10.1016/j.jbc.2026.111200

Figure Lengend Snippet: Differences in TAB1 and FAK recognition patterns by Src . A – C , COS-7 cells were transfected with EGFP-tagged TAB1 and Src (WT, Y419F and R178A) and active p38α-CA. D – F , COS-7 cells were transfected with HA-tagged FAK and Src (WT, Y419F and R178A) and active p38α-CA. Cell lysates were immunoblotted with primary antibodies as indicated. The relative quantification of pY481-TAB1/TAB1 and pY576/577-FAK/FAK, respectively, is presented as the mean ± SD of three independent experiments. p values were calculated by Dunnett’s multiple comparisons test was applied. ∗∗ p < 0.01.

Article Snippet: Primary antibodies for p38α (sc-271120), TAB1 (sc-166138), phosphotyrosine (clone PY20, sc-508), GFP (sc-9996), and β-actin (sc-47778) were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Quantitative Proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: Bone morphogenetic protein receptor 2 signaling mediates mitochondrial Ca 2+ transport through its regulation of TAK1 splice variant

doi: 10.1186/s12964-025-02609-x

Figure Lengend Snippet: BMPR2 regulates TAK1-d splice variant to mediate mitochondrial Ca 2+ transport. A Western blot analysis of NSCLC and leukemia cell lines. B Western blot analysis of cells transfected with TAB1 cDNA or control vector after 24 h. C-D Western blot analysis of cells treated with JL189 for ( C ) 2 h or for ( D ) 4 h. E Western blot analysis of Calu-1 cells transfected with BMPR2 and control siRNA for 48 h. Graphs depict changes in the mean phosphorylation of TAK1 Thr184/Thr187 and TAK1 Ser412 expression of 3 independent biological replicates. F Western blot analysis of A549 WT and BMPR2 KO cells. G-H Western blot analysis of cells treated with BMPR1 receptor inhibitors. THP1 cells were treated for 4 h. Graphs represent normalized pTAK1 expression of (G) 3 independent biological replicates and ( H ) 4 independent biological replicates. I Western blot analysis demonstrating TAK1 splice variants using an antibody recognizing the N-terminal end of TAK1. The top is the immunoblot with a short film exposure and bottom immunoblot is with longer film exposure. J Western blot of THP1 cells treated with JL189 for 6 h. Graph represents normalized values of 2 independent biological replicates. K Western blot of cells transfected with TAB1 or control plasmid vectors for 24 h. Graph represents normalized values of 2 independent biological replicates. L-N Flow cytometry analysis of the fluorescence of Rhod-2AM of cells treated with TAK1 inhibitor 5Z-7-OXO 2 µM or 4 µM for 2 h. Graphs represent mean fluorescence of 3 independent biological replicates. O Western blot analysis of cells transfected with control or TAB1 siRNA for 48 h. Graph represents the mean percent change compared to control in normalized pTAK1 expression of 3 independent biological replicates. P-Q Flow cytometry analysis of the fluorescence of Rhod-2AM of cells transfected with TAB1 siRNA or ( M ) TAB1 cDNA. The graphs represent the mean Rhod-2AM fluorescence intensity of 3 (TAB1 siRNA) and 4 (TAB1 cDNA) independent biological replicates. Graphs show standard errors and p values represent the paired Student’s t test, assuming unequal variances

Article Snippet: BMPR2 (Cat. No. SR319490) and TAB1 (Cat. No. SR307092) siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Variant Assay, Western Blot, Transfection, Control, Plasmid Preparation, Phospho-proteomics, Expressing, Flow Cytometry, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Bone morphogenetic protein receptor 2 signaling mediates mitochondrial Ca 2+ transport through its regulation of TAK1 splice variant

doi: 10.1186/s12964-025-02609-x

Figure Lengend Snippet: BMPR2 regulation of mitochondrial Ca 2+ transport through the MCU effects mitochondrial bioenergetics and cell survival. A Western blot analysis of Jurkat and MDA-MB-231 WT and MCU KO cells. B Flow cytometry analysis of the fluorescence of Rhod-2AM of Jurkat WT and MCU KO cells treated with JL189 1.25 µM for 16 h. The graph represents the mean fluorescence of 4 independent biological replicates presented as percent change from its vehicle DMSO control. C Flow cytometry analysis of the fluorescence of Rhod-2AM of MDA-MB-231 WT and MCU KO cells treated with JL189 2.5 µM for 5 h. The graph represents the mean fluorescence of 2 independent biological replicates presented as percent change from its vehicle DMSO control. D-E Cell counts of ( D ) Jurkat and ( E ) MDA-231 WT and MCU KO cells treated with JL189 2.5 µM for 24 and/or 48 h. The graphs represent the mean percent number of dead cells of at least 4 independent biological replicates. F Flow cytometry analysis of the fluorescence of MitoTracker Green of MDA-MB-231 WT and MCU KO cells treated with JL189 2.5 µM for 16 h. The graph shows with mean fluorescence of 5 independent biological replicates presented as percent change from its vehicle DMSO control. G , I Western blot analysis of THP1 and H1299 transfected with TAB1 cDNA for 24 h done in duplicate. Graphs represent normalized TFAM1 and cytochrome b expression as the mean percent change from control of ( G ) 3 and ( I ) 2 independent biological replicates. H , J Western blot of THP1 and H1299 cells treated with 5Z-7-OXO for 2 h. Graphs represent normalized TFAM1 and cytochrome b expression as the mean percent change from control. H 3 independent biological replicates for TFAM1 and cytochrome b. J 3 independent biological replicates for TFAM1 and 2 independent biological replicates for cytochrome b. K Western blot of H1299 cells transfected with control or MCU siRNA for 48 h then treated with 5Z-7-OXO for 2 h. Graph represents the mean normalized TFAM1 expression of 3 independent biological replicates. L Western blot of H1299 tumor xenografts treated with vehicle or JL189 for 3 weeks. Graphs represent normalized values of 6 independent biological replicates. M Western blot analysis of immunoprecipitated (IP) TAB1-Flag or whole cell lysates of Calu-1 cells transfected with insertless cDNA or TAB1-Flag plasmids. Upper immunoblots show short film exposure and lower blot longer film exposure. N-O CellTiter-Glow assay was used to measure ATP levels. N Cells were treated with DMSO or JL189 2.5 µM for 4 h, or 1.5 µM oligomycin A for 50 min. O Cells were treated with DMSO or JL189 2.5 µM for 4 h. Graphs represent 3 independent biological replicates presented as the percent change from control. Graphs show standard errors and p values represent the paired Student’s t test, assuming unequal variances

Article Snippet: BMPR2 (Cat. No. SR319490) and TAB1 (Cat. No. SR307092) siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Western Blot, Flow Cytometry, Fluorescence, Control, Transfection, Expressing, Immunoprecipitation