Journal: Cardiovascular Research

Article Title: The tumour suppressor Ras-association domain family protein 1A (RASSF1A) regulates TNF-α signalling in cardiomyocytes

doi: 10.1093/cvr/cvu111

Figure Lengend Snippet: RASSF1A plays a key role in the recruitment of TRAF2 and TRADD to the TNFRC. ( A ) Isolated adult cardiomyocytes from RASSF1A −/− mice and WT littermates were treated with 10 ng/mL of TNF-α for 30 min. Immunoprecipitation analysis indicated a marked reduction of TRADD and ( B ) TRAF2 co-precipitation with TNFR1 in RASSF1A −/− cardiomyocytes compared with WT ( n = 4 independent animals). ( C ) Immunoprecipitation analysis of neonatal rat cardiomyocytes (NRCMs) treated with 10 ng/mL of TNF-α. Total protein lysates were precipitated using antibodies as indicated in the figure. Western blot analyses showed that RASSF1A co-precipitated with TNFR1, TRADD, and TRAF2. However, RASSF1A did not interact with TNFR2. ( D ) Overexpression of RASSF1A deletion constructs in NRCM using adenoviral constructs. Expression was detected using an anti-Flag antibody and GAPDH as a loading control in total protein lysates. ( E ) Immunoprecipitation analysis showed that only RASSF1A-ΔN was co-precipitated with TRAF2. Both the ΔC and ΔC+RA constructs did not co-precipitate with TRAF2, suggesting that the C-terminal domain of RASSF1A was responsible for binding with TRAF2. ( F ) Similarly, the C-terminal region was also important in mediating interaction with TRADD ( n = 3 independent experiments).

Article Snippet: Plasmid containing human TNFR-associated factor 2 (TRAF2) cDNA was a gift of Dr John Kyriakis (Boston, MA, USA; Addgene plasmid #21586).

Techniques: Isolation, Immunoprecipitation, Western Blot, Over Expression, Construct, Expressing, Control, Binding Assay

Journal: Cardiovascular Research

Article Title: The tumour suppressor Ras-association domain family protein 1A (RASSF1A) regulates TNF-α signalling in cardiomyocytes

doi: 10.1093/cvr/cvu111

Figure Lengend Snippet: RASSF1A gene knockdown using shRNA reduces the formation of TNFRC and the activation of NFκB pathway. ( A ) Western blot analysis showed ablation of RASSF1A expression in NRCM treated with Ad-shRASSF1A. ( B ) Immunoprecipitation experiments suggested a significant reduction in the interaction between TNFR1–TRAF2 and TNFR1–TRADD in NRCM treated with Ad-shRASSF1A. All cells were stimulated with 10 ng/mL of TNF-α. ( C ) Activation of the NFκB pathway was assessed using adenoviral-driven NFκB-luciferase construct. RASSF1A gene inactivation significantly reduced NFκB activation in response to TNF-α induction. This phenotype was restored by overexpression of human RASSF1A (* P < 0.05, n = 3 independent experiments).

Article Snippet: Plasmid containing human TNFR-associated factor 2 (TRAF2) cDNA was a gift of Dr John Kyriakis (Boston, MA, USA; Addgene plasmid #21586).

Techniques: Knockdown, shRNA, Activation Assay, Western Blot, Expressing, Immunoprecipitation, Luciferase, Construct, Over Expression

Journal: Nature Communications

Article Title: Dual membrane receptor degradation via folate receptor targeting chimera

doi: 10.1038/s41467-025-63882-5

Figure Lengend Snippet: a Schematic illustration of Red-Farletuzumab pHrodo to analyze endocytosis comparing cancer versus normal cells. pHrodo is a fluorescent dye with minimal fluorescence at neutral pH but exhibits strong fluorescence in acidic environments following internalization. Both cancer and normal non-tumorigenic cells were incubated with pHrodo Red-Farletuzumab for four hours, and the internalization was assessed using flow cytometry. b Flow cytometry analysis of FRα endocytosis. Cancer cells demonstrated a significantly higher capacity to internalize the pHrodo Red-Farletuzumab conjugates compared to normal non-tumorigenic cells. Data are presented as mean ± SD ( n = 3). The P value was calculated using an unpaired two-sided Student’s t-test. c Schematics of the strategy to understand the trafficking of CAR-GFP mediated by FolTAC and co-localization imaging of CAR-GFP with lysosomal marker-Lamp1b-mCherry. Cells were treated with PBS, Control, or CAR FolTAC at 100 nM for 24 h before performing the imaging. CAR-GFP (green) marks CAR-expressing cells, Lamp1b-mCherry (magenta) labels lysosomes, and Hoechst 33342 (blue) stains nuclei. Merged images highlight subcellular localization and co-localization patterns. Scale bar: 20 μm (white). d Study of the degradation pathway of FolTAC in PC9 cells using the lysosome inhibitor chloroquine (CQ), bafilomycin (Baf), and the proteasome inhibitor MG132. e Competitive rescue of HER2 FolTAC-mediated degradation using FC-Farletuzumab in SKBR3 cells. f Flow cytometry analysis of FOLR1 knockdown SUMP159PT cells. g Western blot analysis of PD-L1 degradation following treatment with PD-L1 FolTAC in SUM159PT FOLR1 knockdown and WT cells. Untreated (0 nM) samples served as the control group for comparison. Quantification analysis of PD-L1 level in SUM159PT FOLR1 knockdown cells treated with or without FolTAC. “UT” denotes untreated controls. Data represent three independent biological replicates and are presented as mean ± SD. Statistical analysis was performed using an unpaired two-sided Student’s t-test, and exact p -values are shown above the figure bars. h Flow cytometry analysis of FRα overexpression in MC38 cells. i , j mPD-L1 degradation mediated by mPD-L1 control ( i ) in MC38, and FolTAC in MC38-WT and MC38- hFOLR1 cells ( j ). k Western blot analysis of VISTA and FRα co-degradation in MC38 cells overexpressed with FRα and VISTA. Created in BioRender. WANG, Z. (2025) https://BioRender.com/d5n9vj6 .

Article Snippet: The mCherry-tagged Rab7 and LAMP1b expression plasmids were obtained from Addgene (Plasmid #221555 and Plasmid #186576, respectively).

Techniques: Fluorescence, Incubation, Flow Cytometry, Imaging, Marker, Control, Expressing, Knockdown, Western Blot, Comparison, Over Expression