Journal: Frontiers in Cellular Neuroscience

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

doi: 10.3389/fncel.2018.00381

Figure Lengend Snippet: Expression levels of proteins that are involved in SOCE in SK-N-SH Htt138Q HAP1A. (A–D) Expression levels of transient receptor potential channel type 1 (TRPC1) (A) , Orai1 (B) , STIM1 (C) and STIM2 (D) proteins (normalized to α-tubulin) in SK-N-SH Htt138Q cotransfected with GFP (black bars) or HAP1A (red bars). On the top of each panel, the representative Western blots are shown. On the bottom of each panel, the expression levels of proteins are plotted as mean ± SEM. * p < 0.05, significant difference in expression level. ns, not significant. The number of tested lysates is shown above the bars.

Article Snippet: For STIM1 detection, the PVDF membrane was treated with the primary monoclonal anti-STIM1 antibody (1:250; catalog no. 610954, BD Bioscience) and peroxidase-conjugated goat anti-mouse IgG heavy-chain constant region secondary antibody (1:30,000; catalog no. A0168, Sigma).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites

doi: 10.1101/2022.04.12.487993

Figure Lengend Snippet: A-B . Confocal images of Immunostaining of endogenous Sec22b using the Synaptic Systems (SYSY) antibody ( A , magenta) or fluorescence of mCherry (mCh)-Sec22b ( B , magenta) in phagocytic MEFs overexpressing GFP-ERGIC-53 (green) and exposed to IgG-beads for 30 min. Sec22b periphagosomal accumulation containing (arrow 1) and devoid (arrow 2 in A, and arrow in B) of the ERGIC marker. Slice 5 of the total stack is shown to highlight total cell staining while Slice 7 is shown in the inset to highlight periphagosomal accumulation. C ) Immunostaining of endogenous Sec22b (green) in phagocytic MEFs exposed to IgG-beads for 30 min with the Santa Cruz antibody (SC). D ) Immunostaining of endogenous Sec22b (white) in phagocytic MEFs overexpressing both the ER-marker RFP-KDEL (magenta) and GFP-ERGIC-53 (green), exposed to IgG-beads for 30 min. Arrow: periphagosomal accumulations of ER marker that are devoid of ERGIC marker and contain a faint but visible Sec22b signal. E ) MEFs overexpressing FCGR2A-GFP (FcR, green) and mCh-Sec22b (magenta) and exposed to IgG-beads for 15 min. F) 3D-CLEM analysis of phagocytic Stim1 -/- ; Stim2 -/- (DKO) MEFs overexpressing mCh-Sec22b (magenta) and YFP-STIM1 (green) exposed to IgG-beads for 30 min. Arrow 1: periphagosomal accumulation of Sec22b co-localizing with STIM1 in an MCS structure. Arrow 2: MCS containing only STIM1 but not Sec22b. One deconvolved confocal slice corresponds approximately to 75 EM slices. Slice 261 of the EM stack is shown in the large images and insets of site 1, while slice 236 is shown for site 2. Insets labelled “a” show an overlay of the EM with the green channel only, those labelled “b” with the red channel only. For A-E bars = 3 µm, for F bars = 1 µm.

Article Snippet: The following antibodies (antibody name/catalog#/dilution) were purchased from: Synaptic Systems (SYSY): rabbit anti-Sec22b (186003/1:1000); Santa Cruz: mouse anti-Sec22b (29-F7) (sc-101267/1:100); Cell Signaling: mouse anti-c-myc antibody (9B11) (2276/1:100), rabbit anti-STIM2 (4917S/1:1000); Thermo: mouse anti-CD16-CD32 (Fc-Block, 14-0161-85, 1:200), rabbit anti-ORP5 (PA5-18221/1:500), mouse-anti-MHC Class I-PE (H-2Kb, AF6-88.5.5.3, 12-5958-80, 1:100) goat-anti-rabbit Alexa Fluor 555 (A21428/1:1000), goat-anti-mouse Alexa Fluor 647 (A21235/1:1000); GeneTex: rabbit anti-ORP8 (GTX121273/1:500); Sigma: mouse anti-α-tubulin (T9026/1:5000), rabbit anti-sheep red blood cell (sRBC, S1389/1:40), mouse anti-FLAG-M2 (F1804/1:1000); mouse anti-GFP (11814460001/1:1000); BD Biosciences: mouse anti-GOK/STIM1 (610954/1:100); Bio-Rad: goat anti-rabbit IgG (H+L) HRP conjugate (170-6515/1:10000), goat anti-mouse IgG (H+L) HRP conjugate (170-6516/1:10000), Innovative Research: human IgG protein A purified (hIgG) (IR-HU-GF); Jackson ImmunoResearch: donkey-anti-mouse Alexa Fluor 488 (715-545-150/1:800), goat-anti-rabbit Alexa Fluor 647 (111-605-003/1:1000).

Techniques: Immunostaining, Fluorescence, Marker, Staining

Journal: bioRxiv

Article Title: The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites

doi: 10.1101/2022.04.12.487993

Figure Lengend Snippet: A) Representative Western blots of SKO ( Stim1 -/- ) and DKO ( Stim1 -/- ; Stim2 -/- ) MEF cell lines stably expressing shCTR and shSec22b incubated with anti-Sec22b (SYSY) and anti-tubulin as control show efficient Sec22b knockdown (quantification in ). B) Representative Western blots of WT, SKO and DKO stable cell lines incubated as above and with anti-STIM1 (left panel) or anti-STIM1 and STIM2 (right panel) confirm the genotype of the cells and show expression of STIM1 is not upregulated upon Sec22b knockdown (quantification in ). C) Related to , quantification of the number (left panel) and kinetics (right panel, t 50% parameter of the Boltzman sigmoidal curve fit) of STIM1 puncta appearing at the TIRF plane after addition of Tg. In WT cells overexpression of Sec22b increases the number of STIM1 puncta, whereas in DKO cells the number is similar but the kinetics are faster. D) Quantification of the kinetics (slope and t 50% parameters of Boltzmann sigmoidal curve fits) of STIM1 puncta arrival at the TIRF plane in response to Tg shows Sec22b knockdown does not impact STIM1 recruitment. Related to . Bar graphs show means +SEM, * p<0.05, **p<0.01.

Article Snippet: The following antibodies (antibody name/catalog#/dilution) were purchased from: Synaptic Systems (SYSY): rabbit anti-Sec22b (186003/1:1000); Santa Cruz: mouse anti-Sec22b (29-F7) (sc-101267/1:100); Cell Signaling: mouse anti-c-myc antibody (9B11) (2276/1:100), rabbit anti-STIM2 (4917S/1:1000); Thermo: mouse anti-CD16-CD32 (Fc-Block, 14-0161-85, 1:200), rabbit anti-ORP5 (PA5-18221/1:500), mouse-anti-MHC Class I-PE (H-2Kb, AF6-88.5.5.3, 12-5958-80, 1:100) goat-anti-rabbit Alexa Fluor 555 (A21428/1:1000), goat-anti-mouse Alexa Fluor 647 (A21235/1:1000); GeneTex: rabbit anti-ORP8 (GTX121273/1:500); Sigma: mouse anti-α-tubulin (T9026/1:5000), rabbit anti-sheep red blood cell (sRBC, S1389/1:40), mouse anti-FLAG-M2 (F1804/1:1000); mouse anti-GFP (11814460001/1:1000); BD Biosciences: mouse anti-GOK/STIM1 (610954/1:100); Bio-Rad: goat anti-rabbit IgG (H+L) HRP conjugate (170-6515/1:10000), goat anti-mouse IgG (H+L) HRP conjugate (170-6516/1:10000), Innovative Research: human IgG protein A purified (hIgG) (IR-HU-GF); Jackson ImmunoResearch: donkey-anti-mouse Alexa Fluor 488 (715-545-150/1:800), goat-anti-rabbit Alexa Fluor 647 (111-605-003/1:1000).

Techniques: Western Blot, Stable Transfection, Expressing, Incubation, Over Expression

Journal: bioRxiv

Article Title: The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites

doi: 10.1101/2022.04.12.487993

Figure Lengend Snippet: A-C) TIRF analysis of wild-type (WT) and DKO MEFs transfected with YFP-STIM1 in combination with ER marker RFP-KDEL or mCh-Sec22b. Cells washed in calcium-free imaging buffer (CF) were imaged every 15 sec, and 1 µM thapsigargin (Tg) was added after 1 min. The number ( B, See also ), size ( C, left panel), and kinetics ( C, right panel) of STIM1 puncta appearance at the TIRF plane, representing ER-plasma membrane (PM) MCS, were quantified 6 min after Tg addition. Larger STIM1 puncta appeared more rapidly upon Sec22b overexpression. In ( A ) error bars are omitted for clarity, mean values and Boltzman sigmoidal curve used to calculate slope parameters are shown. n=8/11;3/5 coverslips WT;DKO KDEL/Sec22b). D) Similar experiments as in A-C revealed Sec22b knockdown in DKO cells does not impact mCh-STIM1 recruitment to ER-PM MCS (n=13/12 shCTR/shSec22b, see also ). E-F) Global store-operated calcium influx was measured using classic Fura-2-based Tg-Calcium re-addition assay (see methods) in WT MEFs transfected with YFP-STIM1 and RFP-KDEL or mCh-Sec22b ( E ) or in WT MEFs transfected only with GFP-KDEL, EGFP-Sec22b, or EGFP-Sec22b-P33 MCS-disrupting mutant ( F ). Influx was faster compared to KDEL and higher compared to wild-type Sec22b in cells expressing the P33 mutant. Error bars are omitted from traces for clarity. G ) Similar measurements of global calcium influx were performed in WT and Stim1 -/- (SKO) cells. Peak influx was higher in WT shSec22b cells (n=15/13;19/20 WT;DKO shCTR/Sec22b). H ) Quantification of Western blots of WT MEF shCTR and shSec22b lysates show no difference in STIM1 expression upon Sec22b knockdown (n=8, blots normalized to anti-tubulin controls, expressed as relative to shCTR, See also ). I ) Quantification of Western blots of SKO and DKO MEF shCTR and shSec22b lysates show robust knockdown (n=5/5 SKO/DKO, blots normalized to anti-tubulin controls, expressed relative to shCTR, see also ). Bar graphs show means +SEM, *p<0.05, *** p<0.001.

Article Snippet: The following antibodies (antibody name/catalog#/dilution) were purchased from: Synaptic Systems (SYSY): rabbit anti-Sec22b (186003/1:1000); Santa Cruz: mouse anti-Sec22b (29-F7) (sc-101267/1:100); Cell Signaling: mouse anti-c-myc antibody (9B11) (2276/1:100), rabbit anti-STIM2 (4917S/1:1000); Thermo: mouse anti-CD16-CD32 (Fc-Block, 14-0161-85, 1:200), rabbit anti-ORP5 (PA5-18221/1:500), mouse-anti-MHC Class I-PE (H-2Kb, AF6-88.5.5.3, 12-5958-80, 1:100) goat-anti-rabbit Alexa Fluor 555 (A21428/1:1000), goat-anti-mouse Alexa Fluor 647 (A21235/1:1000); GeneTex: rabbit anti-ORP8 (GTX121273/1:500); Sigma: mouse anti-α-tubulin (T9026/1:5000), rabbit anti-sheep red blood cell (sRBC, S1389/1:40), mouse anti-FLAG-M2 (F1804/1:1000); mouse anti-GFP (11814460001/1:1000); BD Biosciences: mouse anti-GOK/STIM1 (610954/1:100); Bio-Rad: goat anti-rabbit IgG (H+L) HRP conjugate (170-6515/1:10000), goat anti-mouse IgG (H+L) HRP conjugate (170-6516/1:10000), Innovative Research: human IgG protein A purified (hIgG) (IR-HU-GF); Jackson ImmunoResearch: donkey-anti-mouse Alexa Fluor 488 (715-545-150/1:800), goat-anti-rabbit Alexa Fluor 647 (111-605-003/1:1000).

Techniques: Transfection, Marker, Imaging, Over Expression, Mutagenesis, Expressing, Western Blot

Journal: bioRxiv

Article Title: The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites

doi: 10.1101/2022.04.12.487993

Figure Lengend Snippet: A) Representative Western blots of WT MEFs transfected with siCTR or siSec22b (50 nM) for 48 h incubated with anti-Sec22b (SYSY) and anti-tubulin as control (related to and ). B-C) Representative Western blots of WT MEFs transfected with siCTR (100 nM) or siORP5/8 (50+50 nM) for 48 h incubated with anti-ORP8 (B) or and anti-ORP5 (C) and anti-tubulin as control (related to ). In (B) the two bands correspond roughly to the molecular weight of the canonical (100 kDa, closed arrow) and short (80 kDa) isoform (ORP8s, open arrow). In (C) the band corresponding to the typical running molecular weight of the canonical ORP5 isoform (110 kDa, closed arrow) responded to siRNA treatment while two other bands (presumed non-specific) did not. D) Quantification of Western blots sets represented in A-C shows efficient knockdown for Sec22b and canonical ORP isoforms (n=8;7;4;3 for siCTR;siSec22b;ORP8 in siORP5/8; ORP5 in siORP5/8). E) Confocal images of mCh-STIM1 and shR-EGFP-Sec22b-P33 in shSec22b MEFs exposed to IgG-beads for 30 min (related to ). Arrows: periphagosomal puncta reminiscent of MCS show co-localization of STIM1 and P33. White bar = 3 µm. Bar graphs are means + SEM. *p<0.05, **p<0.01.

Article Snippet: The following antibodies (antibody name/catalog#/dilution) were purchased from: Synaptic Systems (SYSY): rabbit anti-Sec22b (186003/1:1000); Santa Cruz: mouse anti-Sec22b (29-F7) (sc-101267/1:100); Cell Signaling: mouse anti-c-myc antibody (9B11) (2276/1:100), rabbit anti-STIM2 (4917S/1:1000); Thermo: mouse anti-CD16-CD32 (Fc-Block, 14-0161-85, 1:200), rabbit anti-ORP5 (PA5-18221/1:500), mouse-anti-MHC Class I-PE (H-2Kb, AF6-88.5.5.3, 12-5958-80, 1:100) goat-anti-rabbit Alexa Fluor 555 (A21428/1:1000), goat-anti-mouse Alexa Fluor 647 (A21235/1:1000); GeneTex: rabbit anti-ORP8 (GTX121273/1:500); Sigma: mouse anti-α-tubulin (T9026/1:5000), rabbit anti-sheep red blood cell (sRBC, S1389/1:40), mouse anti-FLAG-M2 (F1804/1:1000); mouse anti-GFP (11814460001/1:1000); BD Biosciences: mouse anti-GOK/STIM1 (610954/1:100); Bio-Rad: goat anti-rabbit IgG (H+L) HRP conjugate (170-6515/1:10000), goat anti-mouse IgG (H+L) HRP conjugate (170-6516/1:10000), Innovative Research: human IgG protein A purified (hIgG) (IR-HU-GF); Jackson ImmunoResearch: donkey-anti-mouse Alexa Fluor 488 (715-545-150/1:800), goat-anti-rabbit Alexa Fluor 647 (111-605-003/1:1000).

Techniques: Western Blot, Transfection, Incubation, Molecular Weight

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin.

doi: 10.1159/000495229

Figure Lengend Snippet: Fig. 2. Effect of lithium treatment without and with NFκB inhibitor wogonin on Orai1, STIM1 and STIM2 protein abundance in iPSC-derived neurons from ChAc patients. A,B,C. Original Western blot of (A) Orai1, (B) STIM1 and (C) STIM2 protein abundance in neurons differentiated from iPSCs derived from ChAc patients (ChAc) without or with lithium (24 h, 2 mM) treatment without and with presence of NFκB inhibitor wogonin (50 µM). D,E,F. Arithmetic means (± SEM, n = 5) of (D) Orai1, (E) STIM1 and (F) STIM2 protein abundance in neurons generated from iPSCs derived from ChAc patients without (blue bars) or with (red and grey bars) lithium (24 h, 2 mM) treatment without (red bars) and with (grey bars) presence of NFκB inhibitor wogonin (50 µM). #(p<0.05), ##(p<0.01) indicates statistically significant difference to respective value in the absence of treatment, §(p<0.05), §§§(p<0.001) indicates statistically significant difference to respective value in the absence of wogonin.

Article Snippet: The proteins were separated by 10% SDS-PAGE in Glycine-Tris buffer and electro-transferred onto nitrocellulose membranes for 90 min. After blocking with 5% milk in TBST at room temperature for 1 h, the membranes were incubated with primary anti-ORAI1 antibody (1:1000, Proteintech), anti-STIM1 antibody (1:1000, Cell Signaling), anti-STIM2 antibody (1:1000, Cell Signaling) and anti-GAPDH antibody (1:1000, Cell Signaling) at 4°C overnight.

Techniques: Quantitative Proteomics, Derivative Assay, Western Blot, Generated

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