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