Journal: Journal of Biological Chemistry

Article Title: Heat Shock Protein 27 Functions in Inflammatory Gene Expression and Transforming Growth Factor-β-activated Kinase-1 (TAK1)-mediated Signaling

doi: 10.1074/jbc.m610987200

Figure Lengend Snippet: FIGURE 4. HSP27 regulates IL-1-induced p38 MAPK and JNK activity but not ERK activation. Cells were transfected as indicated and treated with IL-1 for different times. Cells were harvested and lysed. A, p38 MAPK was immunoprecipitated from lysates and assayed for its ability to phosphorylate His-tagged MK2. Graph shows mean p38 MAPK activity from three independent experiments (S.E.) as a percentage of peak activity in Scr-transfected cells. A portion of the lysates was Western-blotted for HSP27 and p38 MAPK. B, JNK was immu- noprecipitated from lysates, and JNK activity was assayed with GST-ATF-2 as a substrate. Graph shows JNK activity (as for A). An aliquot of lysate was analyzed by Western blotting for HSP27 and -tubulin. C, activation of ERK in lysates was detected by Western blotting with an antibody that recognizes ERK phos- phorylated on Thr-185 and Tyr-187. The blot was stripped and re-probed for total ERK. D, cells were transfected with Scr, HSP27#2, or HSP27#5 twice (with a 24-h interval between transfections) and harvested and lysed 24 h after the second transfection. Phosphorylation of p38 MAPK on Thr-180 and Tyr-182, JNK1 on Thr-183 and Tyr-185, JNK2 on Thr-221 and Tyr-223, and ERK (as in C) in lysates was examined by Western blotting with phosphospecific antibodies. p38 MAPK was blotted as a loading control. Data in each panel (A–D) are repre- sentative of at least two independent experiments.

Article Snippet: The membranes were stained with rabbit anti-COX-2 antibody (Alexis), a mouse anti- -tubulin antibody DM-1A (Sigma-Aldrich), a mouse monoclonal antibody against actin (Sigma), a rabbit antiserum raised against MKK3/6 (32), a rabbit antiserum to human HSP27 (7), a rabbit antiserum that recognizes both human HSP27 and murine HSP25 (Stressgen), a rabbit antiserum to the C-terminal peptide of p38 MAPK (33), a rabbit antiserum against TAK1 (sc-7162; Santa Cruz Biotechnology), and a rabbit antiserum against I B (sc-203; Santa Cruz Biotechnology).

Techniques: Activity Assay, Activation Assay, Transfection, Immunoprecipitation, Western Blot, Phospho-proteomics, Control

Journal: Journal of Biological Chemistry

Article Title: Heat Shock Protein 27 Functions in Inflammatory Gene Expression and Transforming Growth Factor-β-activated Kinase-1 (TAK1)-mediated Signaling

doi: 10.1074/jbc.m610987200

Figure Lengend Snippet: FIGURE 5. HSP27 is needed for activation by IL-1 of the p38 MAPK activators (MKK3 and MKK6) and the JNK activators (MKK4 and MKK7). Cells were transfected and treated as before. A, activation of MKK3 and MKK6 was detected with an antibody that recognizes phosphorylation of Ser-189 and Thr-193 of MKK3 and Ser-207 and Thr-211 of MKK6. The blot was stripped and re-probed with an antibody that recognizes total MKK3 and MKK6. B, kinase assay for MKK4 using a GST-JNK2 (K55R) (a mutant form that is incapable of auto- phosphorylation) and Western blots for HSP27 and -tubulin. C, kinase assay for MKK7 (as for B). Similar results were obtained in two separate experiments for each panel.

Article Snippet: The membranes were stained with rabbit anti-COX-2 antibody (Alexis), a mouse anti- -tubulin antibody DM-1A (Sigma-Aldrich), a mouse monoclonal antibody against actin (Sigma), a rabbit antiserum raised against MKK3/6 (32), a rabbit antiserum to human HSP27 (7), a rabbit antiserum that recognizes both human HSP27 and murine HSP25 (Stressgen), a rabbit antiserum to the C-terminal peptide of p38 MAPK (33), a rabbit antiserum against TAK1 (sc-7162; Santa Cruz Biotechnology), and a rabbit antiserum against I B (sc-203; Santa Cruz Biotechnology).

Techniques: Activation Assay, Transfection, Phospho-proteomics, Kinase Assay, Mutagenesis, Western Blot

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: Frontiers in pharmacology

Article Title: A Palmitoylethanolamide Producing Lactobacillus paracasei Improves Clostridium difficile Toxin A-Induced Colitis.

doi: 10.3389/fphar.2021.639728

Figure Lengend Snippet: FIGURE 4 | Prophylactic administration of pNAPE-LP and ultralow palmitate administration on pro-inflammatory signaling molecules expression and release in mice colon. Immunoreactive bands showing TLR-4, RhoA GTP, phosphorylated/unphosphorylated - p38 MAPK, NF-κB-related p50 and p65, and HIF-1α protein expression in both wild type (A) and PPARα KO (B) mice following TcdA challenge and their relative PPARα-dependent decrease following pNAPE-LP/palmitate co- administration. Relative densitometric analysis (C, D) of each protein (arbitrary units normalized on the expression of the housekeeping protein GAPDH). Results were expressed as mean ± SEM of n 6 experiments performed in triplicate. ***p < 0.001 vs. vehicle; and ooop < 0.001 and oop < 0.01 vs. TcdA. The figure also shows the effect of pNAPE-LP on TcdA challenged wild type (E) and PPARα KO mice (F) in terms of release of IL-6, nitric oxide (NO), and VEGF in the plasma. Results were expressed as mean ± SEM of n 6 experiments performed in triplicate. ***p < 0.001 vs. vehicle; and ooop < 0.001 vs. TcdA.

Article Snippet: After the transfer the membranes were incubated with 10% nonfat dry milk in PBS overnight at 4°C and then exposed, depending on the experiments, with rabbit polyclonal anti-NAPE-PLD (Abcam, Cambridge, United Kingdom) (1:200 v/v), rabbit polyclonal anti–toll-like receptor-4 (TLR4) (Bioss Antibodies, Boston, United States) (1:1,000 v/v), mouse monoclonal anti-RhoAGTPase (Santa Cruz Biotechnology, Santa Cruz, CA, United States) (1:100 v/v), rabbit polyclonal anti-p38 mitogen-activated protein kinase (p38 MAPK) (Bioss Antibodies, Boston, United States (1:1,000 v/v), rabbit monoclonal anti-phospho-p-38 (p-p38) MAPK (Santa Cruz Biotechnology, Santa Cruz, CA, United States) (1:1,000 v/v), rabbit polyclonal anti-NF-κB p65 (Sigma-Aldrich, Milan, Italy) (1:1,000 v/v), mouse monoclonal anti-NF-κB p50 (Santa Cruz Biotechnology, Santa Cruz, CA, United States) (1:1,000 v/v), mouse monoclonal anti–hypoxia-inducible factor-1-alpha (HIF-1α) (Novus biological, Abingdon, United Kingdom) (1: 500 v/v), and rabbit polyclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Danvers, MA, United States) (1:1,000 v/v) according to standard experimental protocols.

Techniques: Expressing, Clinical Proteomics