Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin inhibits MAPK signaling pathway through suppressing CRAF and MEK1/2 but not BRAF kinase activity. a , b The inhibitory effect of erianin on the activity of MEK1 and MEK2 kinase. Active GST-MEK1 full length or GST-MEK2 full length (60 ng) and various doses of erianin were incubated with inactive GST-ERK1 or tag free ERK2 (400 ng) as substrate at 30 °C for 30 min. The phosphorylation of ERK1/2 (Thr202/Tyr204) was detected by western blotting. c The inhibitory effect of erianin on the activity of CRAF kinase. Active CRAF (306-end) (50 ng) and various doses of erianin were incubated with inactive GST-MEK1 (600 ng) as substrate at 30 °C for 30 min. d – f Quantifications of integrated density in ( a – c ) were performed. Data were shown as means ± S.D. of three independent experiments. The asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a significant difference in the expression of phosphorylation of ERK1 or ERK2 vs total ERK1 or ERK2 in control and erianin-treated group. g The luminescent ADP detection assay was developed to detect the luminescence signal of ATP-to-ADP using the same concentration kinases and substrates described in above kinase assay. Three independent repeats were conducted in this experiment. h Immunoprecipitation (IP)/WB of endogenous CRAF from lysates of SK-MEL-2 (NRAS mut) and A375 (BRAF V600E) cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for BRAF, CRAF, and MEK1. i IP/WB of endogenous MEK1 from lysates of SK-MEL-2 and A375 cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for CRAF and MEK1. j , k Western blotting of phospho-CRAF, phospho-MEK1/2 and phospho-ERK1/2 by erianin, vemurafenib, cobimetinib or LY3009120 at indicated concentration for 24 h in NRAS mutant SK-MEL-2 and BRAF V600E mutant A375 cell lines. l Western blotting of MAPK signa l ing pathway by erianin, vemurafenib, cobimetinib, or LY3009120 at indicated concentrations for 24 h in KRAS mutant HCT116 cell line

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Activity Assay, Incubation, Phospho-proteomics, Western Blot, Expressing, Control, Detection Assay, Concentration Assay, Kinase Assay, Immunoprecipitation, Mutagenesis

Journal: Molecular cancer

Article Title: A novel polypeptide CAPG-171aa encoded by circCAPG plays a critical role in triple-negative breast cancer.

doi: 10.1186/s12943-023-01806-x

Figure Lengend Snippet: Fig. 6 CAPG-171aa interacts with STK38 activating the downstream MEK1/2-ERK1/2 pathway via MEKK2. (A) MDA-MB-231 and MDA-MB-468 cell lysates were IP with anti-MEKK2 antibody followed by detection with anti-MEKK2, STK38, and SMURF1 antibody. (B) MDA-MB-231 and MDA-MB-468 were trans fected with CAPG-171aa-FLAG. Whole-cell lysates were IP with anti-SMURF1 and IgG antibodies followed by detection with anti-FLAG, STK38, SMURF1, and GAPDH antibodies. (C-D) Before being treated with MG132, MDA-MB-231, and MDA-MB-468 were transfected with CAPG-171aa-FLAG and circCAPG KD plasmids. Ubiquitination and protein expression levels of MEKK2 were assayed in CAPG-171aa OE (C) and circCAPG KD (D) MDA-MB-231 and MDA- MB-468. (E) Ubiquitination and protein expression levels of MEKK2 were assayed in circCAPG KD MDA-MB-231 and MDA-MB-468 through pulse-chase experiments with cycloheximide. (F) IB of MEKK2, p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in circCAPG KD MDA-MB-231 and MDA-MB-468. (G) IB of p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in OE STK38 MDA-MB-231 and MDA-MB-468. (H) IB of MEKK2, p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in CAPG-171aa, OE-STK38 and OE-STK38/CAPG-171aa transfected MDA-MB-231 and MDA-MB-468. All data were representative of at least three biological replicates and shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: MDA-MB-231 and MDA-MB-468 cells were treated with 10 μg/mL MG132, respectively, (Solarbio, IM0310) for 12 h. Cell lysates were obtained using PierceTM IP lysis buffer (Thermo Fisher Scientific, USA) supplemented with a cocktail (Thermo Fisher Scientific, USA) and then incubated with anti-MEKK2 antibody (Proteintech, USA) and Protein A/G beads overnight at 4°C.

Techniques: Transfection, Ubiquitin Proteomics, Expressing, Pulse Chase

Journal: International Journal of Biological Sciences

Article Title: LncRNA SLCO4A1-AS1 predicts poor prognosis and promotes proliferation and metastasis via the EGFR/MAPK pathway in colorectal cancer

doi: 10.7150/ijbs.38041

Figure Lengend Snippet: Proposed schematic model illustrating the role of SLCO4A1-AS1 in regulating CRC by EGFR/MAPK signaling pathway. SLCO4A1-AS1 influences EGFR/MAPK signaling pathway by promoting the expression of EGFR, KRAS, BRAF, MEK, ERK, MAP3K1 and its corresponding phosphorylated protein levels, which further affect the proliferation, migration and invasion of CRC cells.

Article Snippet: The membrane was incubated with primary antibody overnight at 4 °C, including EGFR (1:2000, Abcam), P-EGFR (1:2000, Abcam), KRAS (1:2000, Abcam), BRAF (1:2000, Abcam), MEK1/2 (1:2000, Abcam), P-MEK1/2 (1:1000, Proteintech), ERK (1:2000, Abcam), P-ERK (1:2000, Abcam), MAP3K1 (1:700, Proteintech), P-MAP3K1 (1:1000, Proteintech), β-actin (1:700, Proteintech), incubate for 2 hours at room temperature with anti-rabbit secondary antibody.

Techniques: Expressing, Migration

Journal: Investigative Ophthalmology & Visual Science

Article Title: AIF-1 Drives Corneal Neovascularization by Promoting Inflammatory Macrophage Activation via the MAPK and PI3K/AKT/mTOR Signaling Pathways

doi: 10.1167/iovs.67.2.22

Figure Lengend Snippet: AIF-1 siRNA inhibited the phosphorylation of P38, ERK1/2, JNK, PI3K, AKT, and mTOR proteins in corneal tissues following alkali burn. ( A ) WB analysis of p-P38, p-ERK1/2, and p-JNK expression levels in CNV corneas. ( B – D ) Quantitative analysis of the WB bands ( n = 3). ( E ) The protein expression level of p-PI3K, p-AKT, and p-mTOR in CNV corneas. ( F-H ) Quantitative analysis of the WB bands ( n = 3). β-Actin served as an internal control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are presented as the mean ± SEM.

Article Snippet: The PVDF membrane was incubated overnight at 4°C with primary antibodies, including AIF-1 (1:1000; Abcam, Cambridge, UK), VEGFA (1:1000; CST, Danvers, MA, USA), CD86 (1:1000; Abcam), TNF-α (1:1000; ABclonal, Wuhan, China), IL-1β (1:2000; ABclonal), IL-6 (1:1000; CST), CD31 (1:1000; R&D Systems, Minneapolis, MN, USA), proliferating cell nuclear antigen (PCNA, 1:1000; ABclonal), p-ERK1/2 (1:1500; Proteintech, Wuhan, China), ERK1/2 (1:1500; Proteintech), P38 (1:1000; CST), p-P38 (1:1000; CST), p-JNK (1:1000; CST), JNK (1:1000; CST), p-PI3K (1:500; Affinity Biosciences, Nanjing, China), PI3K (1:1000; Proteintech), p-AKT (1:3000; Proteintech), AKT (1:1000; Proteintech), p-mTOR (1:5000; Proteintech), mTOR (1:1000; Proteintech), and β-actin (1:2000; Elabscience, Wuhan, China).

Techniques: Phospho-proteomics, Expressing, Control

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: FAM65A binds to Ras and activates the Ras/ERK signaling to mediate RSK activation (A) The volcano plot analysis results for the FAM65A high-expression and low-expression groups from the TCGA database were shown. (B) The KEGG and GO results were shown. (C) The GSEA results were shown. (D) GSEA on DEGs between the FAM65A high-expression group and low-expression group in the Reactome database were shown. (E) IP was performed to detect the binding of FAM65A and Ras/p-RSK. (F) IP was performed to detect the binding of Ras and FAM65A/p-RSK. (G) Immunofluorescence was performed to detect the co-localization of FAM65A and Ras. Scale bars, 20 μm. (H) Western blot analysis the Ras and p -ERK expression in FAM65A knockdown or overexpression cells. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Activation Assay, Expressing, Binding Assay, Immunofluorescence, Western Blot, Knockdown, Over Expression

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: Ras/ERK signaling activation was indispensable for FAM65A-mediated RSK activation and CRC progression (A) Western blot analysis of Ras and p -ERK expression in HCT116-FAM65A cells treated with 10 μM Abd-7, or without treatment. (B) Results from the CCK8 cell proliferation assay conducted on HCT116-FAM65A cells with and without the application of Abd-7, n = 3, ∗∗∗ p < 0.001. (C) Colony formation assay performed on HCT116-FAM65A cells treated with Abd-7 or not. (D) Quantitative analysis of the colony formation assay results, n = 3, ∗∗∗ p < 0.001. (E) Results from the EdU assay conducted on HCT116-FAM65A cells with and without the application of Abd-7. Scale bars, 100 μm. (F) Quantitative analysis of the EdU assay results, n = 3, ∗∗∗ p < 0.001. (G) Western blot analysis of Ki-67, cleaved Caspase 3, Bcl-2, and Bax expression in HCT116-FAM65A cells treated with Abd-7 or not. (H) Results from the apoptosis assay conducted on HCT116-FAM65A cells treated with Abd-7 or not. Scale bars, 50 μm. (I) Quantitative analysis of the apoptosis experiments, n = 3, ∗∗∗ p < 0.001. (J) Results from the Transwell migration assay conducted on HCT116-FAM65A cells with and without the application of Abd-7. Scale bars, 50 μm. (K) Quantitative analysis of the Transwell migration assay results, n = 3, ∗∗∗ p < 0.001. (L) Results from the wound healing assay performed on HCT116-FAM65A cells treated with Abd-7 or not. Scale bars, 50 μm. (M) Quantitative analysis of the wound healing assay results, n = 3, ∗∗∗ p < 0.001. (N) Western blot analysis the expression of EMT markers in HCT116-FAM65A cells treated with Abd-7 or not. (O) Proposed model of FAM65A in CRC progression. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Activation Assay, Western Blot, Expressing, Proliferation Assay, Colony Assay, EdU Assay, Apoptosis Assay, Transwell Migration Assay, Wound Healing Assay

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: Knockdown of FAM65A inhibits tumor progression in vivo (A) LOVO-shCtrl and LOVO-shFAM65A cells were administered into the fourth fat pad of nude mice, and the resulting tumor growth curves were subsequently generated, n = 5, ∗ p < 0.05. (B) The tumors excised from mice across various experimental groups are presented. (C) Hematoxylin and Eosin (HE) staining results of lung tissue from the different groups is displayed. (D) A quantitative analysis of metastatic lung nodules is provided, n = 5, ∗∗ p < 0.01. (E) IHC results for FAM65A, Ki-67, p -RSK, p -ERK, Ras, N-cadherin, vimentin, cleaved Caspase 3, ZO-1, and E-cadherin in tumor tissues are illustrated. (F) A quantitative analysis of the IHC results is included. Data are presented as mean ± SEM of biologically independent experiments, n = 5, ∗∗∗ p < 0.001.

Article Snippet: p -ERK , Proteintech , Cat# 28733-1-AP; RRID: AB_2881202.

Techniques: Knockdown, In Vivo, Generated, Staining

Journal: iScience

Article Title: Zhigancao decoction alleviates Parkinson’s disease via inhibiting TNF/NF-κB and Ras/ERK-mediated neuroinflammation and apoptosis

doi: 10.1016/j.isci.2025.114489

Figure Lengend Snippet: Network pharmacology predicted that ZGCD treatment for Parkinson’s disease might be associated with apoptosis, inflammation, TNF/NF-κB, and Ras/ERK signaling pathways (A) ZGCD Herbal Active Ingredient-Target Network. (B) Venn diagrams of active ingredients and disease targets. (C) The PPI network of the common targets of ZGCD and PD. (D) GO enrichment analysis of potential therapeutic targets. (E) Enrichment analysis of KEGG pathways for potential therapeutic targets.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against TH (1:1000, Abcam, USA), α-Syn (1:1000, Abcam, USA), TNFR1 (1:1000, Proteintech, China), P65 (1:1000, Abcam, USA), P-P65 (1:1000, Abcam, USA), Ras (1:3000, Abcam, USA), ERK (1:2000, Proteintech, China), p -ERK (1:2000, Proteintech, China), TNF-α (1:1000, Proteintech, China), Bax (1:1000, Proteintech, China), Bcl-2 (1:1000, Proteintech, China), Caspase-3 (1:1000, Proteintech, China), Cleaved Caspase-3 (1:1000, Proteintech, China), and GAPDH (1:3000, Abcam, USA) ( ).

Techniques: Protein-Protein interactions, Biomarker Discovery

Journal: iScience

Article Title: Zhigancao decoction alleviates Parkinson’s disease via inhibiting TNF/NF-κB and Ras/ERK-mediated neuroinflammation and apoptosis

doi: 10.1016/j.isci.2025.114489

Figure Lengend Snippet: ZGCD treatment may alleviate MPTP-induced injury of SN DA neurons by inhibiting the TNF/NF-kB and Ras/ERK pathways (A) WB was used to detect the representative expressions of TNFR1, P65, and p-P65 in SN. (B and C) Statistics of the relative expression level of TNFR1 and p-P65/P65 in SN ( n = 4). (D) WB was used to detect the representative expressions of Ras, ERK, and p -ERK in SN. (E and F) Statistics of the relative expression level of Ras and p -ERK/ERK in SN ( n = 4). (G–K) Representative expression of Tnfr1, Nfkb, Grb2, Ras, and Erk detected by RT-qPCR ( n = 4). ### p < 0.001 vs. Con; n.s., not significant, ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 vs. MPTP. Data are expressed as mean ± SD.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against TH (1:1000, Abcam, USA), α-Syn (1:1000, Abcam, USA), TNFR1 (1:1000, Proteintech, China), P65 (1:1000, Abcam, USA), P-P65 (1:1000, Abcam, USA), Ras (1:3000, Abcam, USA), ERK (1:2000, Proteintech, China), p -ERK (1:2000, Proteintech, China), TNF-α (1:1000, Proteintech, China), Bax (1:1000, Proteintech, China), Bcl-2 (1:1000, Proteintech, China), Caspase-3 (1:1000, Proteintech, China), Cleaved Caspase-3 (1:1000, Proteintech, China), and GAPDH (1:3000, Abcam, USA) ( ).

Techniques: Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: Zhigancao decoction alleviates Parkinson’s disease via inhibiting TNF/NF-κB and Ras/ERK-mediated neuroinflammation and apoptosis

doi: 10.1016/j.isci.2025.114489

Figure Lengend Snippet: ZGCDS treatment may alleviate MPTP-induced injury of SN DA neurons by inhibiting the TNF/NF-kB and Ras/ERK pathways (A–C) The levels of TNF-α, IL-1β, and IL-6 were detected by ELISA in SH-SY5Y cells ( n = 3). (D) WB was used to detect the representative expressions of TNF-α in SH-SY5Y cells. (E) Statistics of the relative expression level of TNF-α in SH-SY5Y cells ( n = 3). (F–H) Representative expression of TNF-α, IL-1β, and IL-6 detected by RT-qPCR ( n = 3). (I–N) IF was used to analyze the expressions of TNF-α, IL-1β, and IL-6 in SH-SY5Y cells ( n = 3). Scale bars, 100 μm. (O–Q) Representative expression of Bax, Bcl-2, and caspase-3 detected by RT-qPCR in SH-SY5Y cells ( n = 3). (R–T) WB analysis of Bax, Bcl-2, Caspase-3, and Cleaved Caspase-3 expression in SH-SY5Y cells ( n = 3). ### p < 0.001 vs. Con; n.s., not significant, ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 vs. MPTP. Data are expressed as mean ± SD.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against TH (1:1000, Abcam, USA), α-Syn (1:1000, Abcam, USA), TNFR1 (1:1000, Proteintech, China), P65 (1:1000, Abcam, USA), P-P65 (1:1000, Abcam, USA), Ras (1:3000, Abcam, USA), ERK (1:2000, Proteintech, China), p -ERK (1:2000, Proteintech, China), TNF-α (1:1000, Proteintech, China), Bax (1:1000, Proteintech, China), Bcl-2 (1:1000, Proteintech, China), Caspase-3 (1:1000, Proteintech, China), Cleaved Caspase-3 (1:1000, Proteintech, China), and GAPDH (1:3000, Abcam, USA) ( ).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: Zhigancao decoction alleviates Parkinson’s disease via inhibiting TNF/NF-κB and Ras/ERK-mediated neuroinflammation and apoptosis

doi: 10.1016/j.isci.2025.114489

Figure Lengend Snippet: ZGCDS exerts neuroprotective effects by inhibiting the activation of TNF/NF-κB and Ras/ERK signaling pathways in SH-SY5Y cells (A) WB was used to detect the representative expressions of TNFR1, P65, and p-P65 in SH-SY5Y cells. (B) Statistics of the relative expression level of TNFR1 in SH-SY5Y cells ( n = 3). (C) Statistics of the relative expression level of p-P65/P65 in SH-SY5Y cells ( n = 3). (D–G) IF was used to analyze the expressions of TNFR1 and p-P65 in SH-SY5Y cells ( n = 3). Scale bars, 200 μm. (H–J) WB analysis of Ras and ERK expression and phosphorylation in SH-SY5Y cells ( n = 6). (K–P) IF was used to analyze the expressions of Grb2, Ras, and p -ERK in SH-SY5Y cells ( n = 3). Scale bars, 100 μm. (Q–S) WB analysis of TNFR1 and P65 expression and phosphorylation in SH-SY5Y cells ( n = 4). (T–W) WB analysis of Grb2, Ras, and ERK expression and phosphorylation in SH-SY5Y cells ( n = 4). ### p < 0.001 vs. Con; n.s., not significant, ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 vs. MPTP. Data are expressed as mean ± SD.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against TH (1:1000, Abcam, USA), α-Syn (1:1000, Abcam, USA), TNFR1 (1:1000, Proteintech, China), P65 (1:1000, Abcam, USA), P-P65 (1:1000, Abcam, USA), Ras (1:3000, Abcam, USA), ERK (1:2000, Proteintech, China), p -ERK (1:2000, Proteintech, China), TNF-α (1:1000, Proteintech, China), Bax (1:1000, Proteintech, China), Bcl-2 (1:1000, Proteintech, China), Caspase-3 (1:1000, Proteintech, China), Cleaved Caspase-3 (1:1000, Proteintech, China), and GAPDH (1:3000, Abcam, USA) ( ).

Techniques: Activation Assay, Protein-Protein interactions, Expressing, Phospho-proteomics

Journal: iScience

Article Title: Zhigancao decoction alleviates Parkinson’s disease via inhibiting TNF/NF-κB and Ras/ERK-mediated neuroinflammation and apoptosis

doi: 10.1016/j.isci.2025.114489

Figure Lengend Snippet: Bioactive compounds (Apigenin, Bisdemethoxycurcumin) from ZGCD may exert anti-PD effects by targeting the TNF/NF-kB and Ras/ERK pathways (A and B) Total ion chromatograms (TICs) of Con in positive (A) and negative (B) ion modes. (C and D) Total ion chromatograms (TICs) of ZGCD-CS in positive (C) and negative (D) ion modes. (E) Venn diagram of serum pharmacochemical analysis. (F) Molecular docking analysis of Apigenin and Bisdemethoxycurcumin with key proteins in the pathway. (G–I) The levels of TNF-α, IL-1β, and IL-6 were detected by ELISA in SH-SY5Y cells ( n = 3). (J–L) WB analysis of TNFR1 and P65 expression and phosphorylation in SH-SY5Y cells ( n = 3). (M–P) WB analysis of Grb2, Ras, and ERK expression and phosphorylation in SH-SY5Y cells ( n = 3). ### p < 0.001 vs. Con; n.s., not significant, ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 vs. MPTP. Data are expressed as mean ± SD.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against TH (1:1000, Abcam, USA), α-Syn (1:1000, Abcam, USA), TNFR1 (1:1000, Proteintech, China), P65 (1:1000, Abcam, USA), P-P65 (1:1000, Abcam, USA), Ras (1:3000, Abcam, USA), ERK (1:2000, Proteintech, China), p -ERK (1:2000, Proteintech, China), TNF-α (1:1000, Proteintech, China), Bax (1:1000, Proteintech, China), Bcl-2 (1:1000, Proteintech, China), Caspase-3 (1:1000, Proteintech, China), Cleaved Caspase-3 (1:1000, Proteintech, China), and GAPDH (1:3000, Abcam, USA) ( ).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Phospho-proteomics