v5 tag e9h8o  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: Cytoplasmic connexin43-microtubule interactions promote glioblastoma stem-like cell maintenance and tumorigenicity

doi: 10.1038/s41419-025-07514-2

Figure Lengend Snippet: A Cellular thermal shift assay in VTC-037 GSC lysates was used to determine JM2 and JM2-scrambled selective target engagement potency for α-tubulin and β-tubulin. VTC-037 GSC lysates were subjected to different concentration of JM2 or JM2-scrambled (JM2-scrbl) at 57 °C, and peptide affinity was analyzed by western blotting using antibodies against α-tubulin, β-tubulin, and β-actin as negative control. B Percentage of stabilized α-tubulin and β-tubulin at 57 °C was represented, and EC50 values for JM2 and JM2-scrambled (JM2-scrbl) were calculated. C STORM derived point-cloud localizations of Cx43 (green) and α-tubulin (magenta) in VTC-037 GSCs following treatment with JM2-scrambled (JM2-scrbl) or JM2 at 50 μM for 24 h. Zoomed out panels (left) scale bar: 6 μm. Zoomed in panels (middle) scale bar: 1 μm. Zoomed in panels (right) scale bar: 1 μm. Sphere size: 50 nm. D Cross-Pair correlation functions for Cx43/α-tubulin interaction in C ( n = 10). E VTC-037 GSCs were treated or not with antennapedia (ant), JM2, or JM2-scrambled (JM2-scrbl) at 50 μM for 24 h. Following cross-linking, cell lysates were subjected to co-immunoprecipitation using α-tubulin antibody, or V5 antibody for negative control, and immunoblotted using antibodies against Cx43, α-tubulin, and GAPDH for loading control. Neutravidin-HRP was used to detect biotin-tagged peptides. HC heavy chain, LC light chain.

Article Snippet: Mouse-IgG (R&D SystemsTM; MAB002), mouse antibodies against HA.11 tag (Biolegend; 901513), or V5 tag (Cell Signaling Technology; 80076) were used as isotype negative controls.

Techniques: Thermal Shift Assay, Drug discovery, Concentration Assay, Western Blot, Negative Control, Derivative Assay, Immunoprecipitation, Control

Journal: The EMBO Journal

Article Title: Autophagosomes anchor an AKAP11-dependent regulatory checkpoint that shapes neuronal PKA signaling

doi: 10.1038/s44318-025-00436-x

Figure Lengend Snippet: ( A ) Immunoblot of whole cell lysate from HEK293T sgNT and sgAKAP11 and sgAKAP11 cells stably expressing the indicated V5-AKAP11 construct in triplicate. Cells were grown in DMEM + 10% dFBS. These samples were used for phosphoproteomic experiments. ( B ) Western blots of sgRIα lysates used in Kemptide kinase assay showing vinculin as a loading control and transient expression of FLAG-RIα rescue constructs.

Article Snippet: FLAG (#14793), HA (#3724) PKA RI-α (D54D9) (#5675), PKA C-α (D38C6) (#5842), LC3B (D11) (#3868), Vinculin (E1E9V) (#13901), TAX1BP1 (D1D5)(#13901), V5 rabbit (#13202), V5 mouse (#80076), SQSTM1/p62 (#397749), PP2AA (#2041), HOP (#5670), eEF1A (#2551), CACYBP (#3354), HSP90 (#4874), CCT2 (#3561), PSMA2 (#11864), PP2AC (#2259), GPI (#57893), GSK3 β (#12456), Synapsin (#4297), Oct4 (#27505) from Cell Signaling Technologies.

Techniques: Western Blot, Stable Transfection, Expressing, Construct, Kinase Assay, Control

Journal: Science advances

Article Title: Retromer promotes the lysosomal turnover of mtDNA.

doi: 10.1126/sciadv.adr6415

Figure Lengend Snippet: Fig. 1. mtDNA damage follows a lysosomal-dependent degradation pathway. (A) Confocal images of HeLa cells in the steady state and expressing TWNKK319E- mCherry (mtDNA stress) labeled with α-TOM20. (B) mtDNA copy number analysis in HeLa cells transiently transfected with TWNKK319E-mCherry. (C) Schematic representa- tion of the SplitTurboID assay. The early endosomal marker RAB5C and the mitochondrial outer membrane protein SAMM50 were used to determine the proximity proteome of mitochondria and endosomes. Illustration from NIAID NIH BIOART Source. (D and E) Immunostaining of HeLa cells expressing (D) RAB5C-SplitNt-V5 and (E) SAMM50-SplitCt-HA labeled with α-RAB5 and α-V5 and α-TOM20 and α-HA, respectively. (F and G) Pathway enrichment analysis with Metascape showing GO terms for proteins differentially enriched (F) in the steady state and (G) after expression of TWNKK319E-mCherry. (H and I) Volcano plot showing proteins enriched after biotinylation and purification of cells expressing (H) SplitTurboID plasmids and (I) TWNKK319E-mCherry. Differentially expressed proteins compared with cells transduced only with SAMM50-SplitCt-HA (significant: q value < −0.05 and absolute log2 FC > 1) are highlighted in blue (steady state) or red (mtDNA stress) (n = 3 samples per group). P values were calculated using one-way ANOVA with Tukey correction for multiple comparisons. Scale bars, 10 μm. Data are presented as means ± SEM.

Article Snippet: Antibodies used for immunofluorescence were as follows: polyclonal α- TOM20 (11802- 1- AP; 1:1000) and α- VPS26A (12804- 1- AP; 1:1000) from Proteintech; monoclonal α- dsDNA (ab27156; 1:1000), α- PDHX (ab110333; 1:500), α- ATP5A (ab14748; 1:500), and polyclonal α- RAB5 (1:500) from Abcam; polyclonal α- LAMP1 (#9091; 1:500), α- V5 (#13202; 1:500), and monoclonal α- V5 (#80076; 1:500) from Cell Signaling; monoclonal α- VPS35 (sc- 374372; 1:500) from Santa Cruz.

Techniques: Expressing, Labeling, Transfection, Marker, Membrane, Immunostaining, Purification