Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Drug Design, Development and Therapy

Article Title: Inhibition of Pre-B Cell Colony Enhancing Factor Reduces Lung Injury in Rats Receiving Cardiopulmonary Bypass

doi: 10.2147/DDDT.S281554

Figure Lengend Snippet: Adenovirus-encoding sh-PBEF reduced the increased phosphorylation of ERK1/2, Akt, and p38MAPK in rat lung tissue. ( A ) Representative blots; ( B ) Phosphorylation of ERK1/2; ( C ) Phosphorylation of AKT; ( D ) Phosphorylation of p38MAPK vs control; Original blots were shown in Supplemental figure 1.*P<0.05; vs LPS, # P<0.05; vs CPB+Blank, @ P<0.05.

Article Snippet: The membrane was incubated with the following antibodies overnight at 4°C: rabbit anti-PBEF (1:2000, 11776-1-AP, Proteintech); rabbit anti-ERK1/2 (1:1000, 16443-1-AP, Proteintech); rabbit anti-p-ERK1/2 (1:500, bs-3016R, Bioss); rabbit anti-p38MAPK (1:1000, bs-0637R, Bioss); rabbit anti-p-p38MAPK (1:1000, bs-5477R, Bioss); rabbit anti-AKT (1:500, ab8805, Abcam); rabbit anti-p-AKT (1:1000, bs-2720R, Bioss); rabbit anti-AQP1 (1:1000, bs-1506R, Bioss); rabbit anti-AQP5 (1:1000, AF5169, Affinity); rabbit anti-ENaC (1:1000, bs-2957R, Bioss); rabbit anti-surfactant protein D (SP) (1:1000, ab220422, Abcam); and mouse monoclonal anti-GAPDH (1:2000, TA-08, ZSBio).

Techniques:

Journal: BMC cell biology

Article Title: Phosphorylation of p68 RNA helicase by p38 MAP kinase contributes to colon cancer cells apoptosis induced by oxaliplatin.

doi: 10.1186/1471-2121-13-27

Figure Lengend Snippet: Figure 2 MAPKPhosphorylation of p68 by p38 MAPK. (A) Threonine phosphorylations of p68 in HCT116 cells that are treated with 20 μM of oxaliplatin for different times are analyzed by immunobloting the p68 that are immunoiprecipitated (IP:p68) from cell lysates using antibody against phorsphor-threonine (IB:14B3). Phosphorylation of p38 MAPK under the same treatment is analyzed by immunoblot of cell lysates using antibody against the phosphorylated p38. Immunoblot of p68 (IB:p68) in the immunoprecipitates indicate the amounts of p68 that are precipitated. Immunoblot of p38 in the cell lysate (IB:p38) indicate the cellular levels of p38, as a loading control. (B) Co-immunoprecipitation of p38 and p68 in the cell extracts of HCT116 cells with/without oxaliplatin treatment (Oxa, +/−20 μM) was analyzed by immunoblot of p68 immunoprecipitates (IP:p68) using antibody against p38 (IB:p38). Immunoblot of p68 (IB:p68) in the immunoprecipitates indicate the amounts of p68 that are precipitated. IP:IgG is the immunoprecipitation using rabbit IgG, serving as a negative control IP. (C) Phosphorylation of recombinant His-p68 or BSA, as a control, by recombinant p38 in the presence of [γ-32P]-ATP is revealed by autoradiography. The amounts of proteins used in the phosphorylation reactions are shown by coomasie blue stains (CBS). (D) Phosphorylation of p68 by exogenous expression of Flag-tagged p38 MAPK, wild type and constitutively active mutant D176A-F327L, in HCT116 cells are analyzed by immunoblotting the p68 that are immunoiprecipitated (IP:p68) from cell lysates using antibody against phorsphor-threonine (IB:14B3). Immunoblot of p68 (IB:p68) in immunoprecipitates indicate the amounts of p68 that are precipitated. Immunoblot of Flag-tag (IB:FLAG) indicate the exogenous p38 levels. Immunoblot of GAPDH (IB:GAPDH) is a loading control.

Article Snippet: P38α cDNA (Origene) was subcloned into p3XFLAGmyc-CMVTM-24 Expression Vector (Sigma).

Techniques: Western Blot, Phospho-proteomics, Control, Immunoprecipitation, Negative Control, Recombinant, Autoradiography, Expressing, Mutagenesis, FLAG-tag

Journal: BMC cell biology

Article Title: Phosphorylation of p68 RNA helicase by p38 MAP kinase contributes to colon cancer cells apoptosis induced by oxaliplatin.

doi: 10.1186/1471-2121-13-27

Figure Lengend Snippet: Figure 3 Phosphorylation site(s) of p68 by p38 MAPK. (A) Prediction of potential p38 MAPK phosphorylation site(s) in the p68 reading frame and compared to the consensus p38 MAPK phosphorylation sites of several authentic p38 MAPK substrates by a web-based phosphorylation site prediction program NetPhos 2.0, (B) Phosphorylation of recombinant His-p68 and mutants with single site mutation (Upper) and double site mutations (Lower) by recombinant p38 in the presence of [γ-32P]-ATP is revealed by autoradiography. The amounts of proteins used in the phosphorylation reactions are shown by coomasie blue stains (CBS). (C) Phosphorylation of exogenously expressed HA-p68s, wild type (WT) and mutants (Single site mutation, Upper, and Double site mutations, Lower), in HCT116 cells with/without oxaliplatin treatment (Oxa, +/−) are analyzed by immunoblotting the p68 that is immunoiprecipitated (IP:p68) from cell lysates using antibody against phorsphor-theronine (IB:14B3). Immunoblot of p68 (IB:p68) in immunoprecipitates indicate the amounts of p68 that are precipitated.

Article Snippet: P38α cDNA (Origene) was subcloned into p3XFLAGmyc-CMVTM-24 Expression Vector (Sigma).

Techniques: Phospho-proteomics, Recombinant, Mutagenesis, Autoradiography, Western Blot

Journal: Antioxidants

Article Title: Differences in Airway Remodeling and Emphysematous Lesions between Rats Exposed to Smoke from New-Type and Conventional Tobacco Varieties

doi: 10.3390/antiox13050511

Figure Lengend Snippet: Cigarette smoke-altered Nrf2 and p38 MAPK expression in the lung tissues of rats. Immunohistochemical staining of Nrf2 ( A – D ), p38 MAPK ( E – H ) and phospho-p38MAPK ( I – L ). Representative images at 60 days. 200×, scale bars = 50 µm. The relative expression levels of Nrf2 ( M ), p38 MAPK ( N ) and phosphor-p38 MAPK ( O ). The ratio of phosphor-p38 to p38 ( P ). Results are presented as means with standard deviations, as shown by the vertical bars (n = 6). The statistical significance between all data is compared. Bars with the same letter(s) indicate no significant differences at the level of p < 0.05.

Article Snippet: Rabbit polyclonal antibodies against Bcl-2 (26593-1-AP), Nrf2 (16396-1-AP), p38 MAPK (14064-1-AP) and phospho-p38 MAPK (Thr180/Tyr182) (28796-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Autophagy

Article Title: Macrophage autophagy protects against liver fibrosis in mice

doi: 10.1080/15548627.2015.1058473

Figure Lengend Snippet: ROS generation and activation of MAPK14 is associated with enhanced IL1A/B production by macrophages from atg5−/− mice. (A) ROS production in peritoneal macrophages isolated from atg5−/− or WT mice and exposed to 10 ng/ml LPS. Data are the mean ± SEM (n = 4). &, p < 0.05 for WT vs atg5−/− (B) Il1a and pro-il1b mRNA expression and production in peritoneal macrophages isolated from atg5−/− or WT mice and exposed to 10 mM NAC or its vehicle for 1 h and further stimulated with 10 ng/ml LPS for 6 h. Data are the mean ± SEM from sextuplate repeats. *, p < 0.05 for NAC vs vehicle and &, p < 0.05 for WT vs atg5−/−. (C) Representative P-MAPK14 protein expression by immunocytochemistry in peritoneal macrophages exposed for 15, 30 or 60 min to 10 ng/ml LPS or its vehicle. (D) P-MAPK14 and MAPK14 expression by western blotting in peritoneal macrophages exposed for 15 or 30 min to 10 ng/ml LPS or its vehicle. *, p < 0.05 for WT vs atg5−/− (E) Il1a and pro-il1b mRNA expression in peritoneal macrophages isolated from atg5−/− or WT mice and exposed to 10 μM SB203580 for 1 h and further stimulated with 10 ng/ml LPS for 6 h. Data are the mean ± SEM from sextuplate repeats. *, p < 0.05 for vehicle vs SB203580, &, p < 0.05 for WT vs atg5−/−.

Article Snippet: Cells were fixed in methanol, incubated in a blocking buffer containing 1% BSA and 0.2% Triton X-100 (Sigma, T8787), followed by incubation with an anti-LC3 antibody (1:200, clone 5F10; Nanotools, 0231–100), an anti-SQSTM1/p62 antibody (1:100; ProGen, GP62-C), an anti-ADGRE1 antibody (Serotec, MCA497G) or an anti-P-MAPK14 antibody (1:50; Cell Signaling Technology, 4631).

Techniques: Activation Assay, Isolation, Expressing, Immunocytochemistry, Western Blot