Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: The MK2 kinase activity is not involved, but the MK2 C-terminus is important. ( a ) The rescued MK2 kinase-inactive mutant, MK2K79R, reduces Il1b mRNA levels to a degree comparable to that of the rescued MK2 in untreated (UT) and ( b ) IL-1α-treated (5 ng/mL, 1 h) i MK2 -KO macrophages. ( c ) i MK2 -KO cells have lower levels of the p38α protein. These levels can be restored by rescuing MK2 , but not by rescuing a MK2 mutant lacking the C-terminus MK2-Δ365–386 . ( d ) MK2-Δ365-386 does not affect the Il1b mRNA levels in IL-1α-treated cells. 1W-ANOVA with Tukey’s Multiple Comparison Test, mean ± SEM, ** p < 0.01, *** p < 0.001.

Article Snippet: The n -values of BMDMs of the same genotype differ in certain experiments because there were not enough cells available for some mice to perform all assays in parallel, and some experiments were performed at a different time with other mice of the same genotype. p38α -KO cells were generated by transfecting RAW 264.7 cells ( Mus musculus , CVCL_0493, ATCC) with either the p38α MAPK14 Double Nickase Plasmid (sc-424051-NIC) or the Control Double Nickase Plasmid (sc-437281), according to the Santa Cruz Biotechnology (SCBT, Dallas, TX, USA) protocol.

Techniques: Activity Assay, Mutagenesis, Comparison

Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: p38α inactivates the non-canonical NF-κB pathway independent of the kinase activity. ( a – c ) Overexpression of p38α in i MK2 -KO cells increases basal TRAF2 and reduces basal RelB protein levels. ( d ) Overexpression of p38α and kinase inactive mutant p38-AGF reduce basal Il1b and ( e ) Map3k14 mRNA and ( f ) increase basal Traf2 mRNA in resting i MK2 -KO cells. ( g ) Relb and ( h ) Nfkb2 mRNA are reduced in IL-1α-treated (5 ng/mL, 1 h) i MK2 -KO+ p38α and +p38-AGF cells. ( b , c ) Student’s t -test, ( d – h ) 1W-ANOVA followed by Tukey’s Multiple Comparison Test, mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The n -values of BMDMs of the same genotype differ in certain experiments because there were not enough cells available for some mice to perform all assays in parallel, and some experiments were performed at a different time with other mice of the same genotype. p38α -KO cells were generated by transfecting RAW 264.7 cells ( Mus musculus , CVCL_0493, ATCC) with either the p38α MAPK14 Double Nickase Plasmid (sc-424051-NIC) or the Control Double Nickase Plasmid (sc-437281), according to the Santa Cruz Biotechnology (SCBT, Dallas, TX, USA) protocol.

Techniques: Activity Assay, Over Expression, Mutagenesis, Comparison

Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: Resting p38α -KO cells have elevated levels of non-canonical NF-κB pathway components. ( a ) Resting p38α -KO RAW cells harbor increased Map3k14 mRNA, ( b ) Relb mRNA, and ( c ) Nfkb2 mRNA levels. ( d , e ) Resting p38α -KO RAW cells have elevated RelB and NF κB2 protein levels. 1W-ANOVA followed by Dunnett’s Multiple Comparison Test, mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The n -values of BMDMs of the same genotype differ in certain experiments because there were not enough cells available for some mice to perform all assays in parallel, and some experiments were performed at a different time with other mice of the same genotype. p38α -KO cells were generated by transfecting RAW 264.7 cells ( Mus musculus , CVCL_0493, ATCC) with either the p38α MAPK14 Double Nickase Plasmid (sc-424051-NIC) or the Control Double Nickase Plasmid (sc-437281), according to the Santa Cruz Biotechnology (SCBT, Dallas, TX, USA) protocol.

Techniques: Comparison

Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: MK2/p38α stabilize p53 protein. ( a , b ) Resting i MK2 -KO + GFP cells have less p53 protein level in the nucleus fraction, but similar MDM2 protein level in the cytoplasmic fraction compared to i MK2 -KO + MK2 macrophages. ( c ) TP53 mRNA is increased in i MK2 -KO cells. ( d , e ) Resting p38α -KO RAW macrophages have lower levels of p53 protein in the nuclear fraction, but similar levels of MDM2 protein in the cytoplasmic fraction compared to the control cells. ( f ) TP53 mRNA is increased in p38α -KO cells. ( g , i ) Effect of the translation inhibitor cycloheximide (CHX, 40 µg/mL, 5 h) and the p38 inhibitor BIRB796 (1 µM, 5 h) on the levels of the p53 and RelB proteins in the nuclear fraction of RAW 264.1 cells. ( a – c ) Student’s t -test, ( d – i ) 1W-ANOVA followed by Dunnett’s Multiple Comparison Test, mean ± SEM * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The n -values of BMDMs of the same genotype differ in certain experiments because there were not enough cells available for some mice to perform all assays in parallel, and some experiments were performed at a different time with other mice of the same genotype. p38α -KO cells were generated by transfecting RAW 264.7 cells ( Mus musculus , CVCL_0493, ATCC) with either the p38α MAPK14 Double Nickase Plasmid (sc-424051-NIC) or the Control Double Nickase Plasmid (sc-437281), according to the Santa Cruz Biotechnology (SCBT, Dallas, TX, USA) protocol.

Techniques: Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: MK2/p38/p53 Suppress Basal IL-1β and Non-Canonical NF-κB Signaling in Macrophages

doi: 10.3390/ijms27073232

Figure Lengend Snippet: p53 inactivates the non-canonical NF-κB pathway through RelB cleavage. ( a , b ) Nutlin-3 (20 µM, 4 h) treated i MK2 -KO cells accumulate p53 and harbor reduced RelB protein, but not NF-κB2, TRAF2 or p38α. ( c ) RelB cleavage products (arrows) appear in Western blots after Nutlin-3 (20 µM) treatment in i MK2 -KO cells. ( d ) Il1b mRNA and ( e ) TP53 mRNA are reduced in Nutlin-3 (20 µM)-treated i MK2 -KO cells. ( b ) Student’s t -test, ( d , e ) n = 4, 2W RM-ANOVA with Bonferroni posttests, mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The n -values of BMDMs of the same genotype differ in certain experiments because there were not enough cells available for some mice to perform all assays in parallel, and some experiments were performed at a different time with other mice of the same genotype. p38α -KO cells were generated by transfecting RAW 264.7 cells ( Mus musculus , CVCL_0493, ATCC) with either the p38α MAPK14 Double Nickase Plasmid (sc-424051-NIC) or the Control Double Nickase Plasmid (sc-437281), according to the Santa Cruz Biotechnology (SCBT, Dallas, TX, USA) protocol.

Techniques: Western Blot

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