Journal: iScience

Article Title: Mulberroside A alleviates osteoarthritis via restoring impaired autophagy and suppressing MAPK/NF-κB/PI3K-AKT-mTOR signaling pathways

doi: 10.1016/j.isci.2023.105936

Figure Lengend Snippet: MA inhibits the activation of MAPK and NF-κB signaling pathways in IL-1β-induced chondrocytes Chondrocytes were pretreated with MA (40 μM) for 24 h, then stimulated with or without IL-1β (5 ng/mL) for 15 min. (A) Western blot and (B) quantitative analysis showed the activation of MAPK (JNK, ERK, and P38) and NF-κB (P65) signaling pathway induced by IL-1β could be inhibited by the administration of MA (40 μM). (C) Immunofluorescence staining and (D) quantitative analysis of the nuclear translocation of p-P65 in chondrocytes, scalebar = 50 μm. Data were presented as means ± SD (n = 3). ns, no significance; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Article Snippet: p-P38 , Cell Signaling Technology, Beverly, MA, USA , #4511.

Techniques: Activation Assay, Western Blot, Immunofluorescence, Staining, Translocation Assay

Journal: iScience

Article Title: Mulberroside A alleviates osteoarthritis via restoring impaired autophagy and suppressing MAPK/NF-κB/PI3K-AKT-mTOR signaling pathways

doi: 10.1016/j.isci.2023.105936

Figure Lengend Snippet:

Article Snippet: p-P38 , Cell Signaling Technology, Beverly, MA, USA , #4511.

Techniques: Recombinant, Isolation, Lysis, CCK-8 Assay, Staining, Software

Journal: Experimental and Therapeutic Medicine

Article Title: Salidroside protects against intestinal barrier dysfunction in septic mice by regulating IL‑17 to block the NF‑κB and p38 MAPK signaling pathways

doi: 10.3892/etm.2023.11788

Figure Lengend Snippet: Salidroside affects the activation of NF-κB and p38 MAPK pathways in a septic model. (A) The NF-κB and (B) p38 MAPK signaling pathways activation was detected by ELISA. (C) Western blotting was performed to determine the protein expressions of (D) NF-κB p65 and (E) p38 MAPK. GAPDH was served as the internal reference. The data are presented as the mean ± standard deviation of three independent experiments; *** P<0.001 vs. sham; # P<0.05; ## P<0.01; ### P<0.001 vs. model; ^^ P<0.01; ^^^ P<0.001 vs. salidroside (320 mg/kg).

Article Snippet: The primary antibodies were as follows: NF-κB p65 (Abcam; Rabbit; 1:1,000 dilution; cat. no. ab207297; 65 kDa), phosphorylated (p)-NF-κB p65 (CST; Rabbit; 1:1,000 dilution; cat. no. 3033; 65 kDa), zonula occludens-1 (ZO-1; Abcam; Rabbit; 1:1,000 dilution; cat. no. ab216880; 245 kDa) and claudin-5 (Abcam; Rabbit; 1:1,000 dilution; cat. no. ab13125; 23 kDa), occluding (CST; Rabbit; 1:1,000 dilution; cat. no. 91131; 65 kDa), p38 MAPK (CST; Rabbit; 1:1,000 dilution; cat. no. 8690; 40 kDa), p-p38 MAPK (CST; Rabbit; 1:1,000 dilution; #4511, 43 kDa), Bcl-2 (Abcam; Rabbit, 1:2,000 dilution; cat. no. ab182858; 26 kDa), Bax (Abcam; Rabbit; 1:1,000; cat. no. ab32503; 21 kDa), cleaved-caspase-3 (CST; Rabbit; 1:1,000 dilution; cat. no. 9664; 17 kDa) and GAPDH (Abcam, Mouse, 1:5,000 dilution; cat. no. ab8245; 36 kDa).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Salidroside protects against intestinal barrier dysfunction in septic mice by regulating IL‑17 to block the NF‑κB and p38 MAPK signaling pathways

doi: 10.3892/etm.2023.11788

Figure Lengend Snippet: Anti-IL-17A further affects the activated NF-κB and p38 MAPK pathways in sepsis mice treated with salidroside. The activation of (A) NF-κB and (B) p38 MAPK signaling pathways was examined by ELISA. (C) Western blotting was performed to detect the (D) p-NF-κB p65/NF-κB p65 and (E) -p38 MAPK/p38 MAPK protein expressions and GAPDH was served as the internal reference. The data were presented as the mean ± standard deviation of three independent experiments; *** P<0.001 vs. sham; ## P<0.01 vs. model; ^ P<0.05; ^^ P<0.01 vs. salidroside (160 mg/kg), ^^^ P<0.001; § P<0.05; §§ P<0.01 vs. model + anti-IL-17A, §§§ P<0.001..

Article Snippet: The primary antibodies were as follows: NF-κB p65 (Abcam; Rabbit; 1:1,000 dilution; cat. no. ab207297; 65 kDa), phosphorylated (p)-NF-κB p65 (CST; Rabbit; 1:1,000 dilution; cat. no. 3033; 65 kDa), zonula occludens-1 (ZO-1; Abcam; Rabbit; 1:1,000 dilution; cat. no. ab216880; 245 kDa) and claudin-5 (Abcam; Rabbit; 1:1,000 dilution; cat. no. ab13125; 23 kDa), occluding (CST; Rabbit; 1:1,000 dilution; cat. no. 91131; 65 kDa), p38 MAPK (CST; Rabbit; 1:1,000 dilution; cat. no. 8690; 40 kDa), p-p38 MAPK (CST; Rabbit; 1:1,000 dilution; #4511, 43 kDa), Bcl-2 (Abcam; Rabbit, 1:2,000 dilution; cat. no. ab182858; 26 kDa), Bax (Abcam; Rabbit; 1:1,000; cat. no. ab32503; 21 kDa), cleaved-caspase-3 (CST; Rabbit; 1:1,000 dilution; cat. no. 9664; 17 kDa) and GAPDH (Abcam, Mouse, 1:5,000 dilution; cat. no. ab8245; 36 kDa).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation

Journal: Antioxidants

Article Title: Protopanaxadiol-Enriched Rice Extracts Suppressed Oxidative and Melanogenic Activities in Melan-a Cells

doi: 10.3390/antiox12010166

Figure Lengend Snippet: Effects of the transgenic rice seed extracts on the MAPKs (ERK 1/2 and p38) and PI3K/Akt signaling pathways. DJ, #8, and GE concentrations were 100 µg/mL. S-PPD and DMSO concentrations were 700 pg/mL and 0.1%, respectively. Data are shown as means ± standard deviations. Lowercase letters indicate significant differences among treatments at p < 0.05.

Article Snippet: The transferred protein was incubated with antibodies specific to the melanogenesis-related proteins (microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), TRP-2, and tyrosinase) (Santa Cruz Biotechnology, Dallas, TX, USA), mitogen-activated protein kinases (MAPKs: p38 MAPK, phosphorylation (p)-p38 MAPK, extracellular signal-regulated kinase (ERK), and p-ERK) (Cell Signaling, Danvers, MA, USA), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt and p-Akt; Cell Signaling, Danvers, MA, USA) signaling pathways.

Techniques: Transgenic Assay

Journal: Antioxidants

Article Title: Protopanaxadiol-Enriched Rice Extracts Suppressed Oxidative and Melanogenic Activities in Melan-a Cells

doi: 10.3390/antiox12010166

Figure Lengend Snippet: Schematic diagram of the activation of p-ERK 1/2, p-Akt, and p-p38 on melanogenesis regulation.

Article Snippet: The transferred protein was incubated with antibodies specific to the melanogenesis-related proteins (microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), TRP-2, and tyrosinase) (Santa Cruz Biotechnology, Dallas, TX, USA), mitogen-activated protein kinases (MAPKs: p38 MAPK, phosphorylation (p)-p38 MAPK, extracellular signal-regulated kinase (ERK), and p-ERK) (Cell Signaling, Danvers, MA, USA), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt and p-Akt; Cell Signaling, Danvers, MA, USA) signaling pathways.

Techniques: Activation Assay

Journal: Open Biology

Article Title: Carbonic anhydrases in development: morphological observations and gene expression profiling in sea urchin embryos exposed to acetazolamide

doi: 10.1098/rsob.220254

Figure Lengend Snippet: WB analysis of total proteins from control (C) and AZ-treated embryos (A), collected at 1, 3, 6 and 24 h after AZ addition, and probed with anti-p-p38, anti-p-ERK, anti-c-jun and anti-tubulin (tub) antibodies. Molecular weight markers are indicated on the left (kDa). Protein levels represented in the histogram below were calculated as fold increase/decrease of AZ samples compared to controls, normalized using tubulin (tub) detected on the same membranes. Each bar represents the mean value (±s.d.) of at least two independent experiments. Values included within the −2/+2 range (light grey) were not considered as significantly changed.

Article Snippet: To detect phospho-p38-MAPK (p-p38), phospho-p44/42 MAPK (p-Erk) and c-jun, control (Ctrl) and AZ-treated embryos were Dounce-homogenized on ice in Cell Lysis Buffer (Cell Signaling Technology), supplemented with a cocktail of protease (Roche Applied Science, Penzberg, Germany) and phosphatase (Sigma, Chemical Co., St Louis, MO, USA) inhibitors.

Techniques: Molecular Weight

Journal: Journal of Orthopaedic Surgery and Research

Article Title: GPR84 potently inhibits osteoclastogenesis and alleviates osteolysis in bone metastasis of colorectal cancer

doi: 10.1186/s13018-022-03473-y

Figure Lengend Snippet: GPR84 prevents osteoclast formation through inhibiting MAPK pathways. A and B BMMs isolated from bone marrow were cultured in CM from MC-38 cells and transfected with GPR84-overexpressing vectors (GPR84 OE) or vectors. The protein levels of ERK, JNK, p38 MAPK and their phosphorylation of these proteins were determined by western blotting assay and the protein levels were normalized to β-actin ( A ) and quantification of the protein levels normalized to β-actin ( B ) ( n = 3). C , D Representative TRAP stain images of BMMs were cultured in CM from MC-38 cells, then the cells were transfected with or without GPR84-overexpressing vectors or blank vectors ( C ) and quantification of osteoclast number ( D ) ( n = 5). Bar represents 50 μm. Significant differences are indicated as * p < 0.05, ** p < 0.01 or *** p < 0.001

Article Snippet: The samples were then incubated with antibodies against GPR84 (Affinity), ERK (Cell Signaling Technology), p-ERK (Thr202/Tyr204) (Cell Signaling Technology), JNK (Cell Signaling Technology), p-JNK (Thr183/Tyr185) (Cell Signaling Technology), p38 MAPK (Cell Signaling Technology), p-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology), STAT1 (Cell Signaling Technology), p-STAT1 (Tyr701) (Cell Signaling Technology), and β-actin (Affinity) followed by incubation with HRP-conjugated IgG (1:5000).

Techniques: Isolation, Cell Culture, Transfection, Western Blot, Staining

Journal: bioRxiv

Article Title: Metastatic infiltration of nervous tissue and periosteal nerve sprouting in multiple myeloma induced bone pain

doi: 10.1101/2022.12.29.522199

Figure Lengend Snippet: (A) Overview of the 347 x 260 µm2 area encompassing laminae I-IV of the dorsal horn in which Iba-1, Pp38 and GFAP were analysed. (B, E, H) Representative image of Iba1 + microglia cells (B), Pp38 + microglia cells (E) or GFAP + astrocytes (H) in the ipsilateral dorsal horn of the spinal cord (white arrowheads). (C, D, F, G) The relative number of Iba-1 + (C, D) or P-p38 (F, G) microglia cells in the ipsilateral dorsal horn of the spinal cord of lumbar regions L1 to L6 was unchanged between MM and sham mice euthanized on day 17 or 24. (I, J) The level of normalized GFAP expression in the dorsal horn of the spinal cord was similar between MM and sham mice euthanized on post-surgical day 17 or 24. L=lumbar. Data are presented as mean ± SEM. Sham n=6-8; MM n=7-8.

Article Snippet: Then, slides were o/n incubated with a rabbit antibody against Iba-1 (Wako Pure Chemical Industries, Ltd., Neuss, Germany), GFAP (DAKO Agilent, Glostrup, Denmark) or P-p38 (Thr180/Tyr182) (Bionordika, Cell Signaling Technology, Herlev, Denmark) at 4°C.

Techniques: Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Tripterin liposome relieves severe acute respiratory syndrome as a potent COVID-19 treatment

doi: 10.1038/s41392-022-01283-6

Figure Lengend Snippet: TP lipo inhibits Delta variant in vitro propagation in Vero E6 cells. Vero E6 cells (5 × 10 4 ) were preincubated with TP lipo for 1 h at 37 °C and then exposed to the Delta variant (MOI = 0.05). a Representative CPE pictures estimated at 72 h postinfection are shown under a microscope. b The percentage of CPE at 24, 48, and 72 h was analyzed from three independent experiments. c qRT-PCR quantification of Delta variant gRNA and sgRNA in infected Vero E6 cells at 24 h postinfection. d qRT-PCR quantification of Delta variant gRNA and sgRNA in supernatants at 72 h postinfection. e Delta variant-infected Vero E6 cells were exposed to Vehicle lipo or a different dose of TP lipo for up to 24 h (MOI = 0.05). Total protein was extracted and subjected to analysis of three phosphorylated protein levels, including p-NF κB p65, p-p38 MAPK, and p-STAT3. Vinculin was used as a loading control. Data represent the mean ± SEM; n = 3. Significance is indicated by: ns no significance; * P ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001

Article Snippet: Next, the membranes were probed overnight at 4 °C with the following primary antibodies: mouse polyclonal anti-p-p38 MAPK (T180/Y182) (Cell Signaling Technology, 1:2000), rabbit polyclonal anti-GAPDH (Santa Cruz Biotechnology, 1:4000), rabbit polyclonal anti-STAT3 (HUABIO, 1:1000), rabbit polyclonal anti-p-STAT3 (Tyr705) (HUABIO, 1:500), rabbit polyclonal anti-p-NF-κB p65 (S536) (Cell Signaling Technology, 1:1000), rabbit polyclonal anti-p38 MAPK (Cell Signaling Technology, 1:2500), rabbit polyclonal anti-NF-κB p65 (Cell Signaling Technology, 1:1000), and mouse polyclonal anti-Vinculin (Sigma, 1:4000).

Techniques: Variant Assay, In Vitro, Microscopy, Quantitative RT-PCR, Infection

Journal: Frontiers in Pharmacology

Article Title: Feasibility and mechanism analysis of Reduning in the prevention of sepsis-induced pulmonary fibrosis

doi: 10.3389/fphar.2022.1079511

Figure Lengend Snippet: Expression of the ERBB2-p38 MAPK pathway in AMs. (A) Flow cytometry was used to isolate AMs. (B–D) The expression levels of the ERBB2-p38 MAPK pathway-related proteins were detected by Western blot. Data are presented as mean ± SD ( n = 3 per group) of the representative data from three independent experiments; **** p < 0.001 and #### p < 0.001. The asterisk (*) represents statistically significant difference between the indicated group and the Con group.

Article Snippet: Anti-p-p38 MAPK (Thr180/Tyr182) antibody, anti-p38 MAPK antibody, and FITC-anti-F4/80 antibody were from Cell Signaling Technology.

Techniques: Expressing, Flow Cytometry, Western Blot