rabbit anti human stim1  (Proteintech)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ORAI1 Limits SARS-CoV-2 Infection by Regulating Tonic Type I IFN Signaling.

doi: 10.4049/jimmunol.2100742

Figure Lengend Snippet: FIGURE 1. Loss of ORAI1 reduces basal Ca21 concentration and abrogates SOCE in HEK293-ACE2 cells. (A) Representative immunoblot showing expression of STIM1 in lysates from control, ORAI1/, or STIM1/ HEK293-ACE2 cells. b-Actin is the loading control. Data are representative of two independent experiments. (B) Representative histograms showing levels of total ORAI1 protein in control, ORAI1/, and STIM1/ HEK293-ACE2 cells after permeabilization and intracellular staining with anti-ORAI1 Ab. The bar graph shows average (± SEM) from three independent experiments. (C) Repre- sentative pseudocolored epifluorescence images (original magnification 200) of indicated cells under resting conditions or at the peak of SOCE. (Bottom) Representative traces showing averaged SOCE from control (39 cells), ORAI1/ (35 cells), and STIM1/ (42 cells) HEK293-ACE2 cells after passive depletion of intracellular Ca21 stores with thapsigargin (TG; 1 mM) in Ca21-free external solution. SOCE was measured after replacing external solution with that containing 2 mM CaCl2. Bar graph on the right shows averaged baseline subtracted ER Ca21 stores (TG) and SOCE (± SEM) from four indepen- dent experiments. (D) Bar graph shows baseline Ca21 levels (as depicted by Fura-2 ratio) ± SEM in indicated cell types upon perfusion with extracellular solution containing either 2 or 20 mM CaCl2. Each dot represents data obtained from an independent experiment. ***p < 0.0005 (two-tailed t test).

Article Snippet: Abs for detection of human STIM1 (5668S) were purchased from Cell Signaling Technologies, Abs for detection of IFNAR1 and IFN-b receptor 1 were purchased from Leinco Technologies (I-400), Abs for detection of ORAI1 (ACC-060) were purchased from Alomone Labs, and Abs for detection of b-actin (sc47778) were obtained from Santa Cruz Biotechnology.

Techniques: Concentration Assay, Western Blot, Expressing, Control, Staining, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ORAI1 Limits SARS-CoV-2 Infection by Regulating Tonic Type I IFN Signaling.

doi: 10.4049/jimmunol.2100742

Figure Lengend Snippet: FIGURE 2. Loss of ORAI1 or STIM1 affects cellular susceptibility to SARS-CoV-2 infection. (A) Representative immunoblot showing expression of SARS-CoV-2 proteins in indicated HEK293-ACE2 cells under mock conditions or postinfection with SARS-CoV-2 at indicated MOIs. b-Actin is the loading control. Bottom bar graph shows densitometry analysis of normalized ratio of SARS-CoV-2 Spike protein to b-actin (± SEM) from three independent experi- ments. (B) Representative epifluorescence images (from two independent experiments, 200 magnification) showing expression of spike protein (green) in control, ORAI1/, or STIM1/ HEK293-ACE2 cells postinfection with SARS-CoV-2 at MOI 1.0. Cells were costained with DAPI for detection of nuclei. ORAI1/ HEK293-ACE2 cells were all detached because of high virus load. (C) Quantitative RT-PCR analysis of viral genome from indicated cell types under mock conditions or postinfection with SARS-CoV-2 at indicated MOI for 5 (left graph) or 20 (right graph) h. Shown is one representative triplicate from two independent experiments. **p < 0.005, ***p < 0.0005 (two-tailed t test).

Article Snippet: Abs for detection of human STIM1 (5668S) were purchased from Cell Signaling Technologies, Abs for detection of IFNAR1 and IFN-b receptor 1 were purchased from Leinco Technologies (I-400), Abs for detection of ORAI1 (ACC-060) were purchased from Alomone Labs, and Abs for detection of b-actin (sc47778) were obtained from Santa Cruz Biotechnology.

Techniques: Infection, Western Blot, Expressing, Control, Virus, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ORAI1 Limits SARS-CoV-2 Infection by Regulating Tonic Type I IFN Signaling.

doi: 10.4049/jimmunol.2100742

Figure Lengend Snippet: FIGURE 3. Loss of STIM1 imparts resistance to SARS-CoV-2 infection by enhancing the IFN-b signaling pathway. (A) Levels of IFN-b in culture super- natants from control or STIM1/ HEK293-ACE2 cells under mock conditions or 20 h postinfection with SARS-CoV-2 at indicted MOIs. (B) Levels of IL-6 in culture supernatants from control or STIM1/ HEK293-ACE2 cells. (C) Representative immunoblot showing expression of STIM1 and IFNAR1 in con- trol HEK293-ACE2 cells or those deleted for both STIM1 and IFNAR1 using CRISPR-Cas9mediated recombination. b-Actin is the loading control. Data are representative of two independent experiments. (D) Levels of IFN-b in culture supernatants from control, STIM1/, or STIM1/ IFNAR/ HEK293- ACE2 cells under resting conditions. (E) Representative immunoblot showing expression of SARS-CoV-2 proteins in indicated HEK293-ACE2 cells under mock conditions or postinfection with SARS-CoV-2 at MOI of 0.1. b-Actin is the loading control. Data are representative of two independent infection experiments. (Right) Quantitative RT-PCR analysis of viral genome from indicated cell types under mock conditions or postinfection with SARS-CoV-2 at MOI 0.1 for 20 h. In bar graphs in panels (A), (B), (B), and (E), representative triplicates from two independent experiments are shown. *p < 0.05, **p < 0.005, ***p < 0.0005 (two-tailed t test). N.S., nonspecific band.

Article Snippet: Abs for detection of human STIM1 (5668S) were purchased from Cell Signaling Technologies, Abs for detection of IFNAR1 and IFN-b receptor 1 were purchased from Leinco Technologies (I-400), Abs for detection of ORAI1 (ACC-060) were purchased from Alomone Labs, and Abs for detection of b-actin (sc47778) were obtained from Santa Cruz Biotechnology.

Techniques: Infection, Control, Western Blot, Expressing, CRISPR, Quantitative RT-PCR, Two Tailed Test

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