Journal: Aging (Albany NY)

Article Title: Infusion of blood from young and old mice modulates amyloid pathology

doi: 10.18632/aging.206319

Figure Lengend Snippet: Proteomic analysis of Tg2576 mice infused with blood from old and young wild type mice. ( A ) Heatmap of quantifiable proteins from the Old Blood and Young Blood groups, showing log fold-change (logFC) in protein expression. ( B ) Venn diagram of differentially expressed proteins in an Old Blood vs. Young Blood groups comparison. ( C ) Canonical pathway analysis of differentially expressed proteins, highlighting enriched signaling pathways with activation z-scores. ( D – F ) Differentially expressed proteins in cAMP-mediated signaling, synaptogenesis signaling, and endocannabinoid neuronal synapse pathways, respectively. Gene names are used. ( G , H ) Representative western blot image and quantitative analysis of expression of CACNA2D2 in brain homogenates. ( I , J ) Representative western blot image and quantitative analysis of expression of BRAF in brain homogenates. ( K , L ) Representative western blot image and quantitative analysis of expression of Syngap1 in brain homogenates. ( M , N ) Representative western blot image and quantitative analysis of expression of MAPK9 in brain homogenates. ( O , P ) Representative western blot image and quantitative analysis of expression of GRK2 in brain homogenates. N = 3/group for proteomic analysis, and n = 4/group for protein validation (random mix of males and females; young donor group: 1–2M/2F; old donor group: 1–2M/2F). Data are expressed as mean ± SEM. Data in ( H ), ( J ), ( L ), ( N ), and ( P ) were analyzed using Student’s t -test. * p < 0.05.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4°C in agitation: 6E10 mouse/IgG1 (Biolegend, San Diego, CA, USA) (1/500), α2δ2 ( Cacna2d2 ) rabbit/IgG (Abcam, Fremont, CA, USA) (1:1000), SynGAP1 rabbit/IgG (Abcam, Fremont, CA, USA), BRAF rabbit/IgG (Proteintech, Rosemont, IL, USA), JNK2 (MAPK9) mouse/IgG (Origene, Rockville, MD, USA), GRK2 mouse/IgG1 (Invitrogen, Carlsbad, CA, USA), and β-Actin mouse/IgG2b (Cellsignal, Danvers, MA, USA).

Techniques: Expressing, Comparison, Protein-Protein interactions, Activation Assay, Western Blot, Biomarker Discovery

Journal: Biochemistry and Biophysics Reports

Article Title: Up-regulation of NGEF via the BRAF V600E /ERK/AP1 pathway enhances invasion and migration abilities of BRAF V600E -mutant thyroid cancer

doi: 10.1016/j.bbrep.2025.102164

Figure Lengend Snippet: NGEF expression is significantly enhanced in BRAF V600E -mutated thyroid cancer compared with BRAF wild-type cancer. ( A ) BRAF V600E -mutated thyroid cancers exhibit high NGEF expression levels, compared with BRAF wild-type tissues or healthy tissue obtained from TCGA. ( B ) RT-qPCR was carried out to determine NGEF mRNA levels in the human thyroid epithelial cell line (Nthyori3-1), BRAF wild-type thyroid cancer cells (TPC-1 and KTC-1) and BRAF V600E -mutant thyroid cancer cells (8305C and K1). ( C ) NGEF expression levels were determined using Western blot analysis. ( D ) RT-qPCR was carried out to assess the impact of BRAF V600E and BRAF wild-type on NGEF levels in NIH3T3 cells. ( E ) NIH3T3 cells were transfected with human BRAF wild-type and BRAF V600E , and Western blot analysis was performed to determine the effects on ERK phosphorylation and NGEF expression. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Membranes were blocked with 5 % skimmed milk/TBS-Tween-20 (TBST) for 1 h at room temperature, and subsequently incubated with the primary antibodies against NGEF (cat. no. 13271-1-AP; ProteinTech Group, Inc.), BRAF (cat. no. 20899-1-AP; ProteinTech Group, Inc.), phosphorylated (p)-ERK (cat. no. 28733-1-AP; ProteinTech Group, Inc.), ERK (cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-c-fos (cat. no. 44-280G; Thermo Fisher Scientific, Inc), c-fos (cat. no. 66590-1-Ig; ProteinTech Group, Inc.), p-c-jun (cat. no. 28891-1-AP; ProteinTech Group, Inc.), c-jun (cat. no. T55290 ; Abmart Pharmaceutical Technology Co., Ltd.), MMP2 (cat. no. 10373-2-AP), MMP9 (cat. no. 10375-2-AP), E-cadherin (cat. no. 20874-1-AP; ProteinTech Group, Inc.), N-cadherin (cat. no. 22018-1-AP; ProteinTech Group, Inc.), Vimentin (cat. no. 10366-1-AP; ProteinTech Group, Inc.), Tubulin (cat. no. 11224-1-AP; ProteinTech Group, Inc.) and GAPDH (cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight.

Techniques: Expressing, Quantitative RT-PCR, Mutagenesis, Western Blot, Transfection, Phospho-proteomics

Journal: Biochemistry and Biophysics Reports

Article Title: Up-regulation of NGEF via the BRAF V600E /ERK/AP1 pathway enhances invasion and migration abilities of BRAF V600E -mutant thyroid cancer

doi: 10.1016/j.bbrep.2025.102164

Figure Lengend Snippet: NGEF promotes the migration and invasion of BRAF V600E -mutant thyroid cancer cells. ( A and B ) RT-qPCR and Western blot analysis were used to determine the effects of NGEF knockdown on protein and mRNA expression. ( C and D ) Overexpression of NGEF in K1 and 8305c cells was confirmed using RT-qPCR and Western blot analysis. ( E ) Wound healing in K1 or 8305c cells was assessed following 24 or 36 h (scale bar, 200 μm). ( F ) Transwell assays were used to determine cell invasion and migration following overexpression or knockdown of NGEF in 8305C and K1 cells (scale bar, 100 μm and 50 μm). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗P < 0.001.

Article Snippet: Membranes were blocked with 5 % skimmed milk/TBS-Tween-20 (TBST) for 1 h at room temperature, and subsequently incubated with the primary antibodies against NGEF (cat. no. 13271-1-AP; ProteinTech Group, Inc.), BRAF (cat. no. 20899-1-AP; ProteinTech Group, Inc.), phosphorylated (p)-ERK (cat. no. 28733-1-AP; ProteinTech Group, Inc.), ERK (cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-c-fos (cat. no. 44-280G; Thermo Fisher Scientific, Inc), c-fos (cat. no. 66590-1-Ig; ProteinTech Group, Inc.), p-c-jun (cat. no. 28891-1-AP; ProteinTech Group, Inc.), c-jun (cat. no. T55290 ; Abmart Pharmaceutical Technology Co., Ltd.), MMP2 (cat. no. 10373-2-AP), MMP9 (cat. no. 10375-2-AP), E-cadherin (cat. no. 20874-1-AP; ProteinTech Group, Inc.), N-cadherin (cat. no. 22018-1-AP; ProteinTech Group, Inc.), Vimentin (cat. no. 10366-1-AP; ProteinTech Group, Inc.), Tubulin (cat. no. 11224-1-AP; ProteinTech Group, Inc.) and GAPDH (cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight.

Techniques: Migration, Mutagenesis, Quantitative RT-PCR, Western Blot, Knockdown, Expressing, Over Expression

Journal: Biochemistry and Biophysics Reports

Article Title: Up-regulation of NGEF via the BRAF V600E /ERK/AP1 pathway enhances invasion and migration abilities of BRAF V600E -mutant thyroid cancer

doi: 10.1016/j.bbrep.2025.102164

Figure Lengend Snippet: NGEF induces EMT at the molecular level in BRAF V600E -mutant thyroid cancer cells. ( A ) Correlation between NGEF and pathway score was analyzed using Spearman’s rank correlation coefficient. ( B ) Western blotting analyses of MMP2, MMP9, Vimentin, E-cadherin and N-cadherin expression in K1 and 8305C cells following NGEF knockdown or overexpression. ( C ) Immunofluorescent staining of EMT markers, E-cadherin and Vimentin (scale bar, 50 μm). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗P < 0.001.

Article Snippet: Membranes were blocked with 5 % skimmed milk/TBS-Tween-20 (TBST) for 1 h at room temperature, and subsequently incubated with the primary antibodies against NGEF (cat. no. 13271-1-AP; ProteinTech Group, Inc.), BRAF (cat. no. 20899-1-AP; ProteinTech Group, Inc.), phosphorylated (p)-ERK (cat. no. 28733-1-AP; ProteinTech Group, Inc.), ERK (cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-c-fos (cat. no. 44-280G; Thermo Fisher Scientific, Inc), c-fos (cat. no. 66590-1-Ig; ProteinTech Group, Inc.), p-c-jun (cat. no. 28891-1-AP; ProteinTech Group, Inc.), c-jun (cat. no. T55290 ; Abmart Pharmaceutical Technology Co., Ltd.), MMP2 (cat. no. 10373-2-AP), MMP9 (cat. no. 10375-2-AP), E-cadherin (cat. no. 20874-1-AP; ProteinTech Group, Inc.), N-cadherin (cat. no. 22018-1-AP; ProteinTech Group, Inc.), Vimentin (cat. no. 10366-1-AP; ProteinTech Group, Inc.), Tubulin (cat. no. 11224-1-AP; ProteinTech Group, Inc.) and GAPDH (cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight.

Techniques: Mutagenesis, Western Blot, Expressing, Knockdown, Over Expression, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: Up-regulation of NGEF via the BRAF V600E /ERK/AP1 pathway enhances invasion and migration abilities of BRAF V600E -mutant thyroid cancer

doi: 10.1016/j.bbrep.2025.102164

Figure Lengend Snippet: BRAF V600E mutation upregulates NGEF expression via ERK/AP-1 pathways. ( A ) Co-expression analysis of AP-1 and the corresponding target gene expression using TCGA database. ( B and C ) 8305C and K1 cells were treated either 1 μM GSK120212, 30 μM T5524, or a combination of both for 24 h, and NGEF mRNA and protein expression levels were determined using RT-qPCR and Western blot analysis. ( D ) Dual-luciferase reporter assays were used to evaluate the effects of AP-1 (c-fos/c-jun) on NGEF promoter activity in HEK293T cells. Error bars represent standard deviations. ∗P < 0.05, ∗∗∗P < 0.001.

Article Snippet: Membranes were blocked with 5 % skimmed milk/TBS-Tween-20 (TBST) for 1 h at room temperature, and subsequently incubated with the primary antibodies against NGEF (cat. no. 13271-1-AP; ProteinTech Group, Inc.), BRAF (cat. no. 20899-1-AP; ProteinTech Group, Inc.), phosphorylated (p)-ERK (cat. no. 28733-1-AP; ProteinTech Group, Inc.), ERK (cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-c-fos (cat. no. 44-280G; Thermo Fisher Scientific, Inc), c-fos (cat. no. 66590-1-Ig; ProteinTech Group, Inc.), p-c-jun (cat. no. 28891-1-AP; ProteinTech Group, Inc.), c-jun (cat. no. T55290 ; Abmart Pharmaceutical Technology Co., Ltd.), MMP2 (cat. no. 10373-2-AP), MMP9 (cat. no. 10375-2-AP), E-cadherin (cat. no. 20874-1-AP; ProteinTech Group, Inc.), N-cadherin (cat. no. 22018-1-AP; ProteinTech Group, Inc.), Vimentin (cat. no. 10366-1-AP; ProteinTech Group, Inc.), Tubulin (cat. no. 11224-1-AP; ProteinTech Group, Inc.) and GAPDH (cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight.

Techniques: Mutagenesis, Expressing, Targeted Gene Expression, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay