293t american type culture collection cells  (ATCC)

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

Article Snippet: 293T (American Type Culture Collection) cells were cultured overnight at 37°C in a humidified atmosphere with 5% CO 2 , and treated with either DMSO (vehicle) or 30 μ M ebastine for 1 h at 37°C.

Techniques: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Kv6.1 and variants influence total Kv2.1 expression. A , Kv2.1 was coexpressed with Kv6.1, Kv6.1[L284P], and Kv6.1[W416C] in 1:1 and 1:3 ratios as indicated, for 72 h in HEK293 cells. The Kv2.1 control group was cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in the transfection. Whole-cell lysates were separated using SDS-PAGE and probed with anti-Kv2.1 and β-actin (loading control) antibodies. All the lanes in the representative blot are from the same blot. B , total expression of Kv2.1 in each group was first normalized to their respective β-actin signal and plotted as normalized to the Kv2.1 control expression in each experiment (n = 5). A repeated measures ANOVA test (on raw density normalized to β-actin) followed by a post hoc paired t test was used to compare experimental groups where appropriate (∗ indicates p < 0.05 relative to Kv2.1 alone and # indicates p < 0.05 relative to the Kv2.1:Kv6.1 condition).

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Expressing, Control, Plasmid Preparation, Transfection, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Kv6.1 expression is diminished by coexpression with Kv2.1. A , Kv6.1 and variants were coexpressed with or without Kv2.1 in a 1:1 ratio as indicated, for 72 h in HEK293 cells. The Kv6.1 and variant control groups were cotransfected with GFP to ensure a constant amount of plasmid DNA in the transfection. Whole-cell lysates were separated by SDS-PAGE and probed using an anti-GFP antibody (Kv6.1 constructs are EGFP-tagged), using β-actin as a loading control. Protein ladder molecular weights are in kDa. B , total expression of Kv6.1 in each group was normalized to the β-actin loading control and is plotted as normalized to the WT Kv6.1 control (expressed alone) expression in the corresponding experiment (n = 4–6). A paired t test was used to compare the expression between experimental groups for Kv6.1 or each variant (∗ and ∗∗∗ indicate p values of p < 0.05 and p < 0.001, respectively).

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Expressing, Variant Assay, Control, Plasmid Preparation, Transfection, SDS Page, Construct

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: HALO-label detection of cell surface Kv2.1 expression. A , schematic of a HALO-Kv2.1: Kv6.1 assembly. Membrane-impermeant Halo dye JF635i (shown in red ) reacts with the HaloTag in the extracellular S1-S2 linker of Kv2.1 at the cell surface. B , cumulative probability of JF635i integrated intensity from individual cells in each transfection condition (five independent experiments). C , log of integrated intensity of the HaloJF635i normalized to the Kv2.1 alone group (max value, 1) and the dsRed alone group (min value, 0), for indicated combinations of Kv2.1 and Kv6.1 variants. A repeated measures ANOVA (on the raw median log JF635i integrated intensity in each experiment) followed by a Tukey’s post hoc test was used to compare experimental groups where appropriate (∗ indicates p < 0.05 relative to HALO-Kv2.1 alone and # indicates p < 0.05 relative to the HALO-Kv2.1:Kv6.1 WT condition). D , HEK293 cells were transfected with combinations of HALO-Kv2.1 and Kv6.1 (WT or variants) as indicated, labeled with membrane-impermeant Halo-JF635i, and cell-by-cell fluorescence intensity was quantified using high content microscopy. The scale bar (in white ) represents 100 μm. E , insets from panel D Halo-JF635i label for Kv2.1 alone and with Kv6.1 variants as indicated. F , exemplar sweeps of HALO-Kv2.1 and Kv6.1 with a −40 mV conditioning pulse to assess inactivation (as done in E ). G , voltage dependence of inactivation of HALO-Kv2.1 with Kv6.1 variants (as measured in F , n = 3–6 per group). H , current density at +30 mV for indicated subunit combinations (1:1 cDNA ratio) after transient transfection in LM cells. One-way ANOVA followed by Tukey post hoc test was used to compare experimental groups, with ∗∗∗ and ∗∗ indicating p < 0.001 and p < 0.01, respectively, n = 6 per group. The control groups in all experiments were cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in the transfection mixture.

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Expressing, Membrane, Transfection, Labeling, Fluorescence, Microscopy, Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Stoichiometric interplay affects expression of Kv6.1 and Kv2.1. A , HEK293 cells were transfected with a constant (200 ng) amount of Kv6.1 ( left ) or Kv6.1[W416C] ( right ), and increasing amounts of Kv2.1 (0, 100 ng, 200 ng, 400 ng, 600 ng, 800 ng) for 72 h, and probed for Kv2.1, Kv6.1, or β-actin, as indicated (n = 3). The groups were cotransfected with varying amounts of soluble GFP to ensure constant plasmid DNA in the transfection mixture. B , Kv2.1 bands were normalized to the Kv2.1 signal from the 1:0.5 (WT Kv6.1:Kv2.1) ratio condition. C , Kv6.1 bands were normalized to the Kv6.1 signal from the 1:0 (Kv6.1:Kv2.1) ratio condition. A paired t test was used to compare Kv6.1 versus Kv6.1[W416C] expression at each ratio (∗ indicates p < 0.05, panel C ). Data in ( B , C ) are expressed as mean ± SEM from three independent experiments.

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Kv2.1 coassembly underlies suppression of Kv6.1[W416C]. A , HEK293 cells were transfected with a constant (200 ng) amount of Kv2.1 and increasing amounts of WT Kv6.1 ( left ) or Kv6.1[W416C] ( right ), (Kv6.1 cDNA amounts: 0, 100 ng, 200 ng, 400 ng, 600 ng, 800 ng) for 72 h, and probed for Kv2.1, Kv6.1, or β-actin, as indicated (n = 3). The groups were cotransfected with soluble GFP wherever applicable to ensure a constant amount of plasmid DNA in the transfection mixture. Protein ladder molecular weights are in kDa. B , Kv2.1 signal density was normalized to Kv2.1 signal from the 1:0 (Kv2.1:Kv6.1) ratio condition. A paired t test was used to compare Kv2.1 coexpressed with either Kv6.1 or Kv6.1[W416C] expression at each ratio (∗ indicates p < 0.05, panel B ). Data are expressed as mean ± SEM from three independent experiments. C , Kv6.1 signal density was normalized to WT Kv6.1 signal from the 1:0.5 (Kv2.1:Kv6.1) ratio condition. Data are expressed as mean ± SEM from three independent experiments. D , schematic diagram indicating stability of Kv2.1 and Kv6.1[W416C] heteromers or homomers of each channel under either excess Kv2.1 or excess Kv6.1[W416C] compared to the other one.

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Coassembly with Kv2.1 underlies Kv6.1 phosphorylation. A , Kv2.1 was coexpressed with Kv6.1 variants in HEK293 cells for 72 h. Lysates were treated with Lambda phosphatase (LP) as indicated, separated by SDS-PAGE and probed with an anti-Kv2.1 antibody (n = 3). B , Kv2.1 and Kv6.1 variants were cotransfected in HEK293 cells in 1:1 ratio. Lysates were treated with LP as indicated, separated by SDS-PAGE, and probed with an anti-GFP antibody (n = 3). Arrows denote shifted mobility of Kv6.1 and Kv6.1[L284P] when coexpressed with Kv2.1. Mobility shifts are not seen for Kv6.1 (or variants) when expressed alone. C , Kv2.1 and Kv6.1 variants were cotransfected in HEK293 cells, in 3:1 (Kv2.1:Kv6.1) ratio. Lysates were treated with LP as indicated, separated by a 100 μM phos-tag SDS-PAGE gel, and probed with an anti-GFP antibody (n = 2 of 3:1 ratio and n = 4 for Kv2.1: Kv6.1 in 1:1 ratio, not shown here). Phosphorylated Kv6.1 and Kv6.1[L284P] bands are highlighted with arrows . A higher exposure is shown in the dotted box for clearer depiction of phosphorylated bands. Anti–β-actin was used as a loading control in panels A and B . The control groups (individual subunits expressed alone) were cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in all experiments.

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Phospho-proteomics, SDS Page, Control, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Kv2.1 biogenesis and gating by candidate disease-linked Kv6.1 variants

doi: 10.1016/j.jbc.2025.110943

Figure Lengend Snippet: Common features of Kv2.2 and Kv2.1 modulation by Kv6.1. A , current density at +30 mV was measured from indicated subunit combinations (1:1 cDNA ratio of Kv2.2 and Kv6.1 WT or Kv6.1[W416C]) after transient transfection in LM cells. Control groups were cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in the transfection mixture in all experiments. One-way ANOVA followed by Tukey post hoc test was used to compare groups (∗ indicates p < 0.05; ∗∗ indicates p < 0.001), n = 6. B , mOrange-Kv2.2 was coexpressed with Kv6.1, Kv6.1[L284P], and Kv6.1[W416C] in 1:1 ratio for 72 h in HEK293 cells. Whole-cell lysates were separated on SDS-PAGE and probed with anti-Kv2.2 and Na + /K + -ATPase (loading control) antibodies. C , Kv2.2 signal in each group from panel B is plotted as normalized to the Kv2.2 control (channel expressed alone) (n = 3). A repeated measures ANOVA test (on raw Kv2.2 density normalized to β-actin) followed by a post hoc paired t test was used to compare experimental groups (∗ indicates p < 0.05 relative to Kv2.2 alone). D , Kv6.1 or variants were expressed alone or with Kv2.2 (1:1 ratio), for 72 h in HEK293 cells. Whole-cell lysates were separated by phos-tag SDS-PAGE and probed using an anti-GFP (β-actin loading control (n = 3)). Arrows indicate the slower migration of phosphorylated forms of Kv6.1 and Kv6.1[L284P] when coexpressed with Kv2.2 (representative blot from three experiments).

Article Snippet: HEK293 cells American type culture collection (ATCC) were used for Western blot and high content microscopy experiments.

Techniques: Transfection, Control, Plasmid Preparation, SDS Page, Migration