Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: The effect of FGL2 expression on survival and the correlation between FGL2 and immune checkpoints. ( A ) The expression of FGL2 in a human liver tissue microarray was detected by mIHC. Representative images were shown. Scale bar, 20 μm. Kaplan-Meier analysis of overall survival ( B ) and progression-free survival ( C ) of HCC patients based on FGL2 expression ( n = 85). The Gene Expression Profiling Interactive Analysis server was used to analyze the correlation between Fgl2 and Pdcd1 (encoding PD1, ( D ), Cd274 (encoding PD-L1, ( E ), Pdcd1lg2 (encoding PD-L2, ( F ), Ctla4 ( G ), Tigit ( H ), Entpd1 (encoding CD39, ( I ), Havcr2 (encoding TIM3, ( J ), Btla ( K ) and Lag3 ( L ) at the mRNA expression level, respectively. R=Pearson’s correlation coefficient

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Expressing, Microarray, Gene Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 knockout inhibited tumor growth and downregulated the expression of PD-L1. ( A ) Hepa1-6 cells were inoculated orthotopically into the left liver lobe of WT and Fgl2 -/- mice. The concentration of FGL2 in tumor tissue homogenate was detected by ELISA ( n = 5). ( B ) The livers from C57BL/6 mice bearing orthotopic tumors were shown ( n = 8). ( C ) Representative immunohistochemical images for PD-L1 staining in orthotopic tumors ( n = 4). ( D ) The expression level of PD-L1 on CD45- tumor cells was measured by flow cytometry ( n = 4). Data were represented as median fluorescence intensity (MFI). ( E ) Hepa1-6 cells were inoculated subcutaneously (s.c.) into the right inguinal fold region of WT and Fgl2 -/-mice. The concentration of FGL2 in tumor tissue homogenate was shown ( n = 5). ( F ) Gross images of s.c. tumors of WT or Fgl2 -/- mice ( n = 8). Tumor volume ( G ) and tumor weight ( H ) of the indicated group of C57BL/6 mice ( n = 8). ( I ) The expression level of PD-L1 on tumor cells in the s.c. transplanted hepatomas was detected by flow cytometry ( n = 5). ( J ) WT and Fgl2 -/- mice bearing s.c. tumors were treated with anti-CD3 neutralizing antibodies (200 µg/mice) or IgG1 isotype control (200 µg/mice). Gross images of s.c. tumors in mice treated as indicated ( n = 6). Tumor volume ( K ) and tumor weight ( L ) of the indicated group ( n = 6). Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Knock-Out, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Flow Cytometry, Fluorescence, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 upregulated PD-L1 expression in HCC cells and attenuated the cytotoxicity of T cells. Protein levels of FGL2 (FLAG) and PD-L1 were analyzed by Western blot (WB) in Huh7 ( A ) and MHCC97H cells ( B ) stably expressing FGL2-FLAG. Lentivirus expressing shRNA against FGL2 (sh-FGL2) was used for FGL2 knockdown, and nontargeting shRNA(sh-control) was added as control. FGL2 and PD-L1 expression in MHCC97H cells were detected by WB ( C ), immunofluorescence (IF) ( D ) and flow cytometry (FCM) ( E ). Scale bar, 25 μm. FGL2 knockdown or nontargeting shRNA-transfected MHCC97H cells were treated with IFN-γ (50 ng/mL) for 24 h. The expression of PD-L1 was detected by WB ( F ) and FCM ( G ). ( H ) T-cell cytotoxicity assay against MHCC97H cells. Apoptotic MHCC97H cells were detected by FCM. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Expressing, Western Blot, Stable Transfection, shRNA, Knockdown, Control, Immunofluorescence, Flow Cytometry, Transfection, Cytotoxicity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 disruption induced lysosome-dependent degradation of PD-L1. HCC cells with FGL2 overexpression ( A ) or knockdown ( B ) were treated with CHX (25 mmol/L) for the indicated time points and then subjected to WB for quantitation of PD-L1 level. The expression of PD-L1 in MHCC97H cells after treatment with proteasome inhibitor (MG132) ( C ) or lysosomal inhibitors, NH4Cl ( D ) and chloroquine ( E ) was detected by WB. ( F ) Images and quantification of LysoTracker red staining in MHCC97H cells were displayed. Scale bar, 100 μm. ( G ) The protein levels of LAMP1 and CTSD were determined by WB. ( H ) The colocalization between PD-L1 and LAMP1 in MHCC97H cells was detected by immunofluorescence assay. The intensity profiles of PD-L1 and LAMP1 along the white line were plotted on the middle panel. The statistical result of the colocalization factor (Pearson’s R value) was shown on the right panel. Scale bar, 25 μm. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Disruption, Over Expression, Knockdown, Quantitation Assay, Expressing, Staining, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 disruption promoted the expression of nuclear TFEB. The cytosolic and nuclear distribution of TFEB ( A ) and MITF ( B ) in MHCC97H cells was detected by WB following FGL2 knockdown. Quantification of nuclear TFEB or MITF to Histone-H3 was shown. ( C ) Images and quantification of nuclear TFEB in MHCC97H cells were displayed. Scale bar, 50 μm. The expression levels of TFEB ( D ) and MITF ( E ) were determined by WB in mouse liver cancer tissues ( n = 6). The transcriptional levels of Lamp1, Atp6v0d2 , Ctns and Ctsb were detected by qPCR in mouse liver cancer tissues ( F ) and MHCC97H cells ( G ). TFEB siRNA or control siRNA was transfected to MHCC97H cells pretreated with FGL2 shRNA or control shRNA. The protein levels of LAMP1, CTSD ( H ), and PD-L1 ( I ) were determined by WB. The relative quantification of LAMP1, CTSD or PD-L1 to GAPDH was shown. ( J ) The expression of TFEB in an HCC tissue microarray was detected by mIHC. Representative images were shown. Scale bar, 20 μm. Kaplan-Meier analysis of the progression-free survival ( K ) and overall survival ( L ) of HCC patients based on nuclear TFEB expression ( n = 85). Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Disruption, Expressing, Knockdown, Control, Transfection, shRNA, Quantitative Proteomics, Microarray

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 interacted with mTOR kinase and affected the phosphorylation of mTORC1 pathway. ( A ) Immunoblot analysis of (p-)mTOR, (p-)P70S6K, (p-)4EBP1 and (p-)AKT in MHCC97H cells following FGL2 knockdown. ( B ) Total P70S6K, 4EBP1 and AKT proteins as well as their phosphorylated forms were detected in liver cancer tissues from WT and Fgl2 -/- mouse(n = 6). ( C ) The phosphorylation level of TFEB Ser211 was detected by WB. The expression levels of (p-)P70S6K, (p-)4EBP1 ( D ) and PD-L1 ( E ) were detected in MHCC97H cells treated with FGL2 shRNA or control shRNA in the absence or presence of mTOR signaling activator (MHY1485). ( F ) Co-immunoprecipitation analysis of the interaction between FGL2 and mTOR in MHCC97H cells stably expressing FGL2-FLAG. ( G ) The colocalization of FGL2 and mTOR in MHCC97H cells was detected by immunofluorescence. Scale bar, 25 μm.( H ) 293T cells were co-transfected with HA-mTOR, FLAG-FGL2 or vector plasmids. Cell lysates were subjected to co-immunoprecipitation using anti-HA or anti-FLAG antibodies. ( I ) 293T cells were co-transfected with HA-mTOR truncations and FLAG-FGL2 plasmids. Cell lysates were subjected to co-immunoprecipitation using anti-FLAG antibodies. ( J ) MHCC97H cell lysates were subjected to immunoprecipitation​ with anti-mTOR antibodies, followed by immunoblot analysis. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Phospho-proteomics, Western Blot, Knockdown, Expressing, shRNA, Control, Immunoprecipitation, Stable Transfection, Immunofluorescence, Transfection, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 inhibition enhanced antitumor immune responses of anti-PD1 therapy in HCC. ( A ) WT and Fgl2 -/- mice bearing s.c. tumors were treated with anti-PD1 monoclonal antibodies (αPD1, 100 µg/mice) or IgG isotype control (100 µg/mice). Gross images of tumors in mice treated as indicated ( n = 6). Tumor volume ( B ) and tumor weight ( C ) of the indicated group. ( D ) Representative immunohistochemical images of subcutaneous tumors for PD-L1 staining ( n = 3). Representative flow cytometric plots and quantification of the proportion of IFN-γ+ CD8+ cells ( E ), granzyme B+ CD8+ cells ( F ) and CD107a+ CD8+ cells ( G ) in tumor tissues in response to different treatments. ( H ) Representative flow cytometric plots and quantification of the proportion of Tregs (CD4+ CD25+ Foxp3+) in tumor tissues from the indicated groups. The proportions of Th1 (CD4+ IFN-γ+ T cells) ( I ) and Th2 (CD4+ IL-4+ T cells) cells ( J ) in tumor tissues were shown. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Inhibition, Bioprocessing, Control, Immunohistochemical staining, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: Schematic of FGL2-mediated PD-L1 regulation and therapy potential of combination interference of FGL2 and PD1. In HCC cells, FGL2 stabilizes the mTOR-Raptor interaction and activates mTORC1 signaling. Activated mTORC1 phosphorylates TFEB, thereby suppressing its nuclear translocation and subsequent lysosomal biogenesis. This lysosomal dysfunction​ attenuates PD-L1 degradation, leading to elevated PD-L1 abundance and the inhibition of T cell–mediated cytotoxicity. In HCC mouse models, FGL2 inhibition exerts synergistic antitumor effects with anti-PD1 therapy by downregulating tumorous expression of PD-L1, promoting the activity of tumor-infiltrating lymphocytes, and suppressing the accumulation of Tregs

Article Snippet: After washing and blocking, the cells were incubated with the following primary antibodies: FGL2 (1:200) (H00010875-M01, Abnova), PD-L1 (1:200) (17952-1-AP, Proteintech), LAMP1 (1:200) (ab25630, Abcam), TFEB (1:200) (13372-1-AP, Proteintech) and mTOR (1:100) (66888-1-Ig, Proteintech).

Techniques: Translocation Assay, Inhibition, Expressing, Activity Assay

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