Journal: Cell reports

Article Title: Altered GM1 catabolism affects NMDAR-mediated Ca 2+ signaling at ER-PM junctions and increases synaptic spine formation in a GM1-gangliosidosis model

doi: 10.1016/j.celrep.2024.114117

Figure Lengend Snippet: (A) Immunoblot analysis of cell fractions from 6-month-old WT and β-Gal − / − mice. Markers for the ER (Calnexin), PM (N-cadherin), and ER-PM junctions (ORAI1, STIM1, STIM2, VAPA, and VAPB) were enriched in their respective fractions. Immunoblots using HRP-conjugated cholera toxin B subunit (CTX-B) show high GM1 levels in β-Gal − / − fractions. (B) Representative HPTLC plate showing GM1 levels in the ER, PM, and ER-PM junctions isolated from 6-month-old WT and β-Gal − / − mice. STD, standard. Note: to detect GM1 in WT samples, the sample volume loaded was 3× that of the β-Gal − / − samples. (C) Quantification of GM1 levels from HPTLC plates performed in (B). n = 8. Values are expressed as median ± quartiles. Statistical analysis was performed using the Student’s t test; *** p < 0.001, **** p < 0.0001. (D) Representative HPTLC plate showing GM1 levels in ER-PM junctions isolated from 1-, 3-, and 6-month-old WT and β-Gal − / − mice. To detect GM1 in WT samples, the sample volume loaded was 3× that of the β-Gal − / − samples. (E) Quantification of GM1 levels from HPTLC plates performed in (D). n = 4. Values are expressed as median ± quartiles. Statistical analysis was performed using the Student’s t test with Welch’s correction; ns, not significant; *** p < 0.001, **** p < 0.0001.

Article Snippet: Rabbit anti-STIM1 antibody , Cell Signaling , Cat #: 4916; RRID: AB_2271287.

Techniques: Western Blot, High Performance Thin Layer Chromatography, Isolation

Journal: Cell reports

Article Title: Altered GM1 catabolism affects NMDAR-mediated Ca 2+ signaling at ER-PM junctions and increases synaptic spine formation in a GM1-gangliosidosis model

doi: 10.1016/j.celrep.2024.114117

Figure Lengend Snippet:

Article Snippet: Rabbit anti-STIM1 antibody , Cell Signaling , Cat #: 4916; RRID: AB_2271287.

Techniques: Virus, Recombinant, Isolation, Magnetic Beads, Plasmid Preparation, Software

Journal: Frontiers in Pharmacology

Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: αPD-1 treatment increases K + channel activity in HNSCC T cells. (A) Representative current traces of KCa3.1 and Kv1.3 channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs cells from a HNSCC patient in absence or presence of αPD-1 (10 μg/ml, for 6 h). Data are normalized to maximum current at +50 mV recorded using a ramp pulse protocol from −120 mV to +50 mV for 200 ms every 15 s. The holding potential used was −70 mV. (B,C) KCa3.1 (B) and Kv1.3 (C) conductance (G) measured in the absence or presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 68 cells without pembrolizumab and n = 55 cells with pembrolizumab from 14 patients). (D) Representative current traces of divalent free current (DVF) through CRAC channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs from a HNSCC patient. Data were recorded using a ramp pulse protocol from −100 to +100 mV with at holding potential of +30 mV every 1.5 s. Cells were perfused with 0 mM Ca 2+ solution (1 min) followed by 20 mM Ca 2+ (1 min) and DVF solutions (2 min, see methods) to amplify currents during recordings. (E) Peak DVF current values measured in absence and presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 34 cells without αPD-1 and n = 31 cells with αPD-1 from 8 patients). The values in panels (B,C) and (E) are represented as box plots: the horizontal line indicates the median; the lower box is the 25 th percentile; the upper box is the 75 th percentile; and the whiskers represent the 10 th and 90 th percentiles. (F) Ion channel expression (KCa3.1, Kv1.3, Orai1 and STIM1) in HNSCC patient T cells after treatment with αPD-1 (10 μg/ml for 6 h). Effect of αPD-1 treatment is shown as ratio of mean fluorescence intensity (MFI, fold change) values of treatment versus control group. Data are represented as scatter plot where each symbol represents an individual patient ( n = 4–5). Horizontal line represents mean values for each group. Data in panels (B,C,E) were analyzed by Mann-Whitney rank sum test.

Article Snippet: Cells were then stained for rabbit anti-human STIM1 (Proteintech) primary antibodies followed by secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher).

Techniques: Activity Assay, Incubation, Expressing, Fluorescence, Control, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.

Article Snippet: Cells were then stained for rabbit anti-human STIM1 (Proteintech) primary antibodies followed by secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher).

Techniques: Inhibition, Patch Clamp, Transferring, Control, Fluorescence, Flow Cytometry

Journal: The Journal of Neuroscience

Article Title: Store-Operated Ca 2+ Entry in Sensory Neurons: Functional Role and the Effect of Painful Nerve Injury

doi: 10.1523/JNEUROSCI.5053-10.2011

Figure Lengend Snippet: Identification of molecular components of store-operated Ca2+ channels. (A.) Western blotting detected Orai1 and STIM1 proteins in lysates from control (C) dorsal root ganglia (DRG), from 4th lumbar DRGs after 5th lumbar spinal nerve ligation (SNL L4), and from axotomized 5th lumbar DRGs after SNL (SNL L5). A dominant STIM1 band at the expected molecular weight is accompanied by a closely coherent secondary band that others have shown to be sensitive to STIM1 knockdown by RNAi (Ong et al., 2007). (Typical of n=3.) (B.) Quantification of Western blots as fold-difference compared to control showed no effect of injury on Orai1 and STIM1 protein levels. mean ± sem. (C.) Quantification of transcript levels by quantitative rtPCR also showed no effects of injury on Orai1 and STIM1 expression. For B. and C., numbers in bars indicate n.

Article Snippet: After blocking with 5% milk in TBST (Tris buffered saline + 0.1% Tween 20), blots were sequentially probed with anti-β-Tubulin I mouse monoclonal antibody (1:20000; Sigma, catalog number T7816), anti-STIM1 rabbit polyclonal antibody (1:500; ProSci, Poway, CA, catalog number 4119), and anti-Orai1 rabbit polyclonal antibody (1:1000; ProSci, catalog number 4281).

Techniques: Western Blot, Control, Ligation, Molecular Weight, Knockdown, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: The Journal of Neuroscience

Article Title: Store-Operated Ca 2+ Entry in Sensory Neurons: Functional Role and the Effect of Painful Nerve Injury

doi: 10.1523/JNEUROSCI.5053-10.2011

Figure Lengend Snippet: Immunohistochemical identification of STIM1 distribution. (A.) Staining for STIM1 (left panels) was uniformly distributed within cytoplasmic areas of cells identified as neuronal profiles through co-staining with neuron-specific nuclear protein (NeuN) (middle panels). Only minimal nonspecific staining was present in fiber tracts. The scale bar applies to each panel.(B.) Morphometric analysis showed comparable intensity (arbitrary units, recorded using standardized image acquisition) of staining in neurons of all sizes (ANOVA P=0.40). mean ± sem. (C.) STIM1 (left panel) was also found in cellular components identified as satellite glial cells by their expression of glutamine synthase (GS, middle panel). Identically placed arrows in the left and middle panels indicate cellular areas clearly identifiable as satellite glial cell cytoplasm that expresses STIM1. The scale bar applies to each panel.

Article Snippet: After blocking with 5% milk in TBST (Tris buffered saline + 0.1% Tween 20), blots were sequentially probed with anti-β-Tubulin I mouse monoclonal antibody (1:20000; Sigma, catalog number T7816), anti-STIM1 rabbit polyclonal antibody (1:500; ProSci, Poway, CA, catalog number 4119), and anti-Orai1 rabbit polyclonal antibody (1:1000; ProSci, catalog number 4281).

Techniques: Immunohistochemical staining, Staining, Expressing