primary antibodies  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Kv1.3 palmitoylation regulates spatial distribution and channel removal from the immunological synapse

doi: 10.64898/2026.01.19.700329

Figure Lengend Snippet: Palmitoylation facilitates Kv1.3 endocytosis. (A) Representative confocal images of HEK293 cells transfected with either WT or Cys less Kv1.3 YFP. The cells were incubated in the absence (-PMA) or presence (+PMA) of PMA for 30 min, after which early endosomes (EEA1) were identified. The plasma membrane (PM) and nuclei were stained with wheat germ agglutinin (WGA) and DAPI, respectively. The merged panels show the indicated colocalizations in white. Green, Kv1.3 channels; magenta, cell markers (EEA1 or PM). Scale bars represent 10 μm. (B) Manders overlapping coefficients between Kv1.3 and EEA1 (top panel) and between Kv1.3 and the PM (bottom panel). Data are presented as the means ± SEs of n > 30 cells from 3 independent experiments. ***p < 0.001 by one-way ANOVA with post hoc Tukey test. (C) Membrane protein biotinylation assay of HEK293 cells transfected with either WT or Cys less Kv1.3 YFP and incubated in the absence (-PMA) or presence (+PMA) of PMA for 30 min to trigger endocytosis. Calnexin was used as a negative control because it is a transmembrane protein located in the ER and is absent at the PM, and β-actin was used as a loading control. (D) Quantification of Kv1.3 biotinylation in arbitrary units (A.U.). The data are presented as the means ± SEs of 4 independent experiments. *p < 0.05 by one-way ANOVA with post hoc Tukey test.

Article Snippet: To study endocytosis, the samples were incubated in blocking solution (5% nonfat milk and 0.5% Triton X-100 in PBS) for 1 h at RT, after which they were incubated with anti-EEA1 primary antibodies (Fisher Scientific) for 2 h at RT.

Techniques: Transfection, Incubation, Clinical Proteomics, Membrane, Staining, Cell Surface Biotinylation Assay, Negative Control, Control

Journal: Journal of Medicinal Chemistry

Article Title: Innately Fluorescent Tetravalent Cytotoxic Conjugate TetraF HER2 -vcMMAE Engages Aggregation-Dependent Endocytosis of HER2 for Enhanced Intracellular Drug Delivery

doi: 10.1021/acs.jmedchem.5c00782

Figure Lengend Snippet: Efficient clustering-based endocytosis of multivalent HER2 ligands. (A) The internalization of BiF HER2 , TriF HER2 , TetraF HER2 , and PentaF HER2 into SKBR-3 cells was analyzed by using quantitative confocal microscopy. Cells were treated with oligomers for 30 min at 37 °C. Nuclei were stained with NucBlue Live dye, and the cells were fixed, permeabilized with 0.1% Triton in PBS, and stained with HCS CellMask Deep Red Stain. Representative images from three independent experiments are shown. The scale bar represents 20 μm. Each gray spot in the graph represents the relative intracellular punctate signal intensity oligomers in the single cell. At least 200 cells for each condition from three independent experiments were measured. Horizontal lines in the graph represent the average intensity of the intracellular oligomer punctate signal, whereas boxes represent ± SD. Statistical analyses were performed using analysis of variance (ANOVA) with Tukey HSD for unequal N (Spjotvoll/Stoline) posthoc test (* p < 0.05; ** p < 0.005 and *** p < 0.001). (B) Colocalization of TetraF HER2 with early endosome marker EEA1. SKBR-3 cells were incubated with TetraF HER2 for 30 min at 37 °C. Early endosomes were detected with rabbit polyclonal antibody specific for early endosome antigen 1 (EEA1) and antirabbit IgG secondary antibody conjugated to Alexa Fluor 594 (red). Scale bars are 20 μm. (C) Colocalization of TetraF HER2 with HER2. SKBR-3 cells were incubated with TetraF HER2 for 30 min at 37 °C. HER2 was detected with mouse monoclonal antibody specific for HER2 (ErbB2/HER2) and antimouse IgG secondary antibody conjugated to Alexa Fluor 594 (red). Scale bars represent 20 μm. (D) DLS signals of TetraF HER2 , HER2, and mixtures of these proteins. DLS-estimated MW of the proteins are shown. High molecular weight complexes are seen upon incubation of TetraF HER2 and HER2. (E) Western blotting analysis of cell lysates of SKBR-3 cells treated with siRNA against clathrin heavy chain (CLTC), dynamin-2 (DNM2), and scramble siRNA as a control. CBB was used as a loading control. (F) Analysis of the effect of the depletion of CLTC and DNM2 on the endocytosis of TetraF HER2 . SKBR-3 cells after CLTC and DNM2 knock-down were incubated with TetraF HER2 for 30 min at 37 °C, and internalization was analyzed using quantitative confocal microscopy. Representative images from three independent experiments are shown. The scale bar represents 20 μm. Each gray spot in the graph represents the relative intracellular punctate signal intensity of the TetraF HER2 in the single cell. At least 200 cells for each condition from three independent experiments were measured. Horizontal lines in the graph represent the average intensity of the intracellular TetraF HER2 punctate signal, whereas boxes represent ± SD. Statistical analyses were performed using analysis of variance (ANOVA) with Tukey HSD for unequal N (Spjotvoll/Stoline) posthoc test (* p < 0.05; ** p < 0.005 and *** p < 0.001).

Article Snippet: Anti-EEA1 primary antibody (no. 2411S) was from Cell Signaling (Danvers, MA).

Techniques: Confocal Microscopy, Staining, Marker, Incubation, High Molecular Weight, Western Blot, Control, Knockdown

Journal: PLOS Pathogens

Article Title: The BICD2 dynein cargo adaptor binds to the HPV16 L2 capsid protein and promotes HPV infection

doi: 10.1371/journal.ppat.1012289

Figure Lengend Snippet: A. HeLa S3 cells were transfected with scrambled control (siScram) or BICD2-targeting (siBICD2) siRNAs and infected with HPV harboring the luciferase reporter plasmid at the MOI of ~200. At 16, 24, and 32 hpi, PLA was performed with antibodies recognizing HPV L1 and EEA1. Mock, uninfected; HPV, infected. PLA signals are green; nuclei are blue (DAPI). Similar results were obtained in two independent experiments. B. The fluorescence of PLA signals was determined from multiple images obtained as in (A). Each dot represents an individual cell (n>40) and black horizontal lines indicate the mean value of the analyzed population in each group. ***p < 0.001; ****p < 0.0001; ns, not significant. The graph shows results of a representative experiment. C. As in (A) except PLA was performed using antibodies recognizing HPV L1 and TGN46. D. As in (B) from multiple images obtained as in (C). **p < 0.01. E. Proposed model for dynein-BICD2 dependent transport of HPV16 during entry. The dynein-BICD2 complex first engages HPV L2 after cytosolic exposure of L2 from the endosome. This interaction helps to transport the virus from the endosome to the TGN/Golgi. The BICD2-dynein complex continues to ferry HPV through the Golgi and eventually to the nucleus for infection. Figure created in BioRender. Blue, BICD2; orange, dynein; red, L2; green, microtubule; light blue, membrane lipid bilayer. BICD2 siRNA #2 was used.

Article Snippet: Primary antibodies recognizing EEA1 (Cell Signaling Technology, 2411) or TGN46 (Abcam, ab50595) were diluted with DMEM10 (1:250) and incubated with the coverslips overnight at 4°C.Secondary antibodies were diluted with TBS containing 0.2% Tween-20 and 3% BSA or DMEM10 and incubated with coverslips for one hour at room temperature.

Techniques: Transfection, Control, Infection, Luciferase, Plasmid Preparation, Fluorescence, Virus, Membrane

Journal: PLOS Pathogens

Article Title: The BICD2 dynein cargo adaptor binds to the HPV16 L2 capsid protein and promotes HPV infection

doi: 10.1371/journal.ppat.1012289

Figure Lengend Snippet: Antibodies and inhibitors.

Article Snippet: Primary antibodies recognizing EEA1 (Cell Signaling Technology, 2411) or TGN46 (Abcam, ab50595) were diluted with DMEM10 (1:250) and incubated with the coverslips overnight at 4°C.Secondary antibodies were diluted with TBS containing 0.2% Tween-20 and 3% BSA or DMEM10 and incubated with coverslips for one hour at room temperature.

Techniques: Solvent