phospho p38 mapk thr180 tyr182 rabbit mab  (Cell Signaling Technology Inc)

Journal: Antibiotics

Article Title: Clarithromycin Modulates Neutrophilic Inflammation Induced by Prevotella intermedia in Human Airway Epithelial Cells

doi: 10.3390/antibiotics13090909

Figure Lengend Snippet: CXCL8 mRNA expression and IL-8 production by P. intermedia supernatants might be induced through transduction via ERK 1/2. ( A ) Inhibition of CXCL8 mRNA expression with dimethyl sulfoxide, Trametinib, Adezmapimod, SP600125, and caffeic acid phenethyl ester at a concentration of 10 μM was analyzed using real-time RT-PCR post 4 h incubation ( n = 4). ( B ) CXCL8 mRNA expression was inhibited by Trametinib pretreatment in a concentration-dependent manner ( n = 4). ( C ) IL-8 production post 24 h incubation was inhibited by Trametinib pretreatment ( n = 4). ( D ) CXCL8 mRNA expression was not influenced by Adezmapimod or SP600125 pretreatment in a concentration-dependent manner ( n = 4). Data are presented as mean +/− SEM, and each figure is representative from two independent experiments. Significant differences are determined using one-way ANOVA, followed by Tukey’s multiple comparisons test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; DMSO, dimethyl sulfoxide; Trametinib, MEK 1/2 inhibitor; Adezmapimod, p38 MAPK inhibitor; SP600125, c-JUN N-terminal kinase inhibitor II; CAPE, NF-kβ inhibitor.

Article Snippet: The following antibodies were supplied by Cell Signaling Technology (Danvers, MA, USA): anti-p44/42 MAPK (Erk1/2) antibody (#9102), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (#9101L), anti-p38 MAPK antibody (#9212), anti-Phospho-p38 MAPK (Thr180/Tyr182) (3D7) rabbit monoclonal antibody (mAb) (#9215), anti-stress-activated protein kinase (SAPK)/JNK antibody (#9252), anti-phospho-SAPK/JNK (Thr183/Tyr185) antibody (#9251), anti-I-kappa-B-alpha (IkBa) (44D4) rabbit mAb (#4812), anti-Phospho-IkBa (Ser32) (14D4) rabbit mAb (#2859), and Erk3 antibody (#4067).

Techniques: Expressing, Transduction, Inhibition, Concentration Assay, Quantitative RT-PCR, Incubation

Journal: bioRxiv

Article Title: A molecular switch for stress-induced activation of retrograde mitochondrial transport

doi: 10.1101/2024.09.13.612963

Figure Lengend Snippet: Representative maximum intensity projection images corresponding to panels E, F, H, I, J are shown in respectively. ( A ) The TRAK2 S84 phosphorylation site identified by mass spectrometry highlighted in an inset showing the CC1-box region of the TRAK2 coiled-coil prediction as in (top). ( B ) S84 conservation in TRAK2. ( C ) Representative maximum intensity projection images of cargo from cell lines expressing wt, S84A or S84E TRAK2. ( D ) Quantification of perinuclear cargo accumulation in cells as shown in (C) from two biological replicates. n(wt)=12; n(S84A)=12; p<0.0001 (two-tailed t-test, t=6.615, df=22); n(S84E)=12; p=0.5889 (two-tailed t-test, t=0.5485, df=22). ( E ) Quantification of perinuclear cargo accumulation in cells expressing synthetic cargo and TRAK2 following mock or JNK-in-8 (10 μM) treatment (3h) from three biological replicates. n(mock)=27; n(JNK-in-8)=27; p<0.0016 (two-tailed t-test, t=3.332, df=52). ( F ) Quantification of perinuclear cargo accumulation in cells expressing TRAK2 following RNAi treatment as indicated from two biological replicates. n(wt)=120; n(JNK1,p38δ)=29, n(JNK2,JNK3)=30; one-way ANOVA (F= 4.271, dFn =4, dFd =233) with a post hoc Dunnett’s test was used to determine significance. ( G ) Immunoblot analysis of untransduced whole cell lysate showing JNK- and p38-kinase activation in response to 3h oxidative treatments as indicated. ( H ) Quantification of mitochondrial clustering following oxidative treatment in untransduced cells from two biological replicates. n(mock)=63; n(arsenite)=18; p<0.0001 (two-tailed t-test, t=6.356, df=113); n(peroxide)=13; p<0.0001 (two-tailed t-test, t=7.832, df=113). ( I ) Quantification of perinuclear cargo accumulation following oxidative treatment in cells expressing TRAK2 from two biological replicates. n(mock)=102; n(arsenite)=13; p<0.0001 (two-tailed t-test, t=6.436, df=79); n(peroxide)=13; p<0.0001 (two-tailed t-test, t=7.336, df=79). ( J ) Quantification of perinuclear cargo accumulation following arsenite treatment in cells expressing wt or S84A TRAK2 from two biological replicates. Arsenite-induced accumulation was normalized to mock treated cells for both variants. n(treated wt)=18; n(treated S84A)=18; p=0.0003 (two-tailed t-test, t=3.996, df=34).

Article Snippet: The following primary antibodies were used: GFP (CST, 2555S), RFP (Proteintech, 6g6), KIF5B (abcam, ab167429), DHC1 (Santa Cruz Biotechnology, sc-514579), p150 (BD Biosciences, 610473), GAPDH (CST, 2118S), DIC (Sigma-Aldrich, MAB1618), p50 (BD Biosciences, 611002), SAPK/JNK (CST, 9252S), Phospho-SAPK/JNK (Thr183/Tyr185) (CST, 9251S), p38 MAPK (CST, 9212S), Phospho-p38 MAPK (Thr180/Tyr182) (CST, 9211S), O-GlcNAc [RL2] (abcam, ab2739), p38δ MAPK (CST, 2308T).

Techniques: Mass Spectrometry, Expressing, Two Tailed Test, Western Blot, Activation Assay