mouse anti stim1  (Proteintech)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A dual topology of STIM1 at the plasma membrane regulates calcium constitutive entry

doi: 10.1007/s00018-026-06141-0

Figure Lengend Snippet: The Endogenous Fraction of STIM1 PM Presents a Dual Topology in Panc-1 Pancreatic Cells. Panel A: STIM1 PM topology in Panc-1-Wt cell plasma membranes was explored by flow cytometry. Permeabilized cells (Panels A1 and A2) or non-permeabilized cells (Panels A3 and A4) were labeled with STIM1 antibodies targeting either the N-terminal region (STIM1 Nter1: GOK Ab coupled to PE—Panels A1 and A3) or the STIM1 C-terminal region (STIM1 Cter3: CDN3H4 Ab coupled to PE—Panels A4) of STIM1. Histograms represent single values of Mean Fluorescence Intensity (MFI) along with the mean MFI value ± SEM detected in cells labeled with STIM1 antibodies ( n ≥ 4 experiments). Line graphs show representative overlays of STIM1 expression in cells labeled with STIM1 antibody (black) or with a control isotype (white) for each experimental condition. Panel B: Topology of STIM1 PM in Panc-1-Wt cell plasma membranes was explored using an ELISA approach. Histograms display single values of optical densities (OD) and the mean OD value ± SEM measured in intact Panc-1-Wt cells labeled with anti-STIM1 antibodies targeting either the N-terminal region (STIM1 Nter1: GOK Ab, n = 8; STIM1 Nter2: ACC-063 Ab, n = 6; and STIM1 Nter3: 4H3 Ab, n = 6—Panel B1) or the C-terminal region (STIM1 Cter1: HPA 011088 Ab, n = 13; STIM1 Cter2: HPA 012123 Ab, n = 8; and STIM1 Cter3: CDN3H4 Ab, n = 9—Panel B2) of STIM1. Panel C: STIM1 PM topology in Panc-1-Wt cell plasma membranes was further confirmed by immunoprecipitating STIM1 PM . STIM1 PM was immunoprecipitated in intact cells with the STIM1 Nter1 Ab (GOK Ab—Panel C1) or the STIM1 Cter3 Ab (CDN3H4 Ab—Panel C2) coupled with G proteins. Representative Western Blots of STIM1 detection performed with either the STIM1 Nter1 Ab (GOK Ab—Panel C1) or the STIM1 Cter3 Ab (CDN3H4 Ab—Panel C2) ( N = 3). Panel D: Schematic representation of STIM1 PM double orientation. Data are analyzed by non-parametric Mann–Whitney test, * P < 0.05 and ** P < 0.01

Article Snippet: Non-specific blocking was done by incubation with 5% fat milk in PBS, 0.1% Tween 20 for 1 h. Blots were incubated overnight with 5% fat milk in PBS, 0.1% Tween 20, containing mouse monoclonal anti-STIM1 (CDN3H4 clone, Santa Cruz; 1:1,000 dilution; sc-66173) or mouse monoclonal anti-STIM1 (GOK BD, Alomone; 1:1,000 dilution; ACC-063) or mouse monoclonal anti-GAPDH antibody (6C5 clone, Abcam; 1:10,000 dilution).

Techniques: Clinical Proteomics, Flow Cytometry, Labeling, Fluorescence, Expressing, Control, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Western Blot, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A dual topology of STIM1 at the plasma membrane regulates calcium constitutive entry

doi: 10.1007/s00018-026-06141-0

Figure Lengend Snippet: STIM1 forms anti-parallel dimers when inserted in the plasma membrane. Panel A: Colocalization of STIM1 PM with N-Ter out -C-Ter in and N-Ter in -C-Ter out orientations in Panc-1-Wt was identified by co-immunofluorescence. Panels A1 and A2 present representative pictures of STIM1 PM localization in non-permeabilized fixed cells, revealed with an anti-STIM1 antibody targeting the N-terminal (anti-STIM1 Nter2, Panel A1) or the C-terminal (anti-STIM1 Cter3, Panel A2) regions of STIM1. Panel A3 presents the co-immunofluorescence obtained when using these two anti-STIM1 antibodies. A picture showing the absence of labeling obtained in cells incubated with an isotype is presented in Panel A4. Pearson Correlation Coefficient (PCC) was measured using Fiji and is represent in the graph ( Panel A5 ) to confirm colocalisation of STIM1 PM N out -C in and N in -C out . Panel B: Assessment of STIM1 PM anti-parallel homodimerization in Panc-1-Wt was confirmed using a proximal ligation assay (PLA) approach. Antibodies targeting either the N-terminal region (anti-STIM1 Nter2) or the C-terminal region (anti-STIM1 Cter3) were used for the simultaneous labeling of STIM1 with both N-Ter out -C-Ter in and N-Ter in -C-Ter out orientations in non-permeabilized and fixed Panc-1-Wt cells. A representative picture showing fluorescent dots corresponding to interacting STIM1 proteins with antiparallel orientations is presented in this panel. Experiments from panels A and B were repeated twice, and no points were detected when cells were labeled with isotypes. An optical 20X magnification was used, and a scale bar of 20 μm is reported on each picture. Panel C: Application of the protein-fragment complementation (PCA) based assay NanoLuc® Binary Technology (NanoBiT) confirmed that STIM1 forms at least antiparallel homodimers when localized at the plasma membrane. Cells were transfected with STIM1-LrgBiT and SmBiT-STIM1 or with MRAP2-LrgBiT and SmBiT-MRAP2 as a positive control, or with RAMP3-LrgBiT and SmBiT-RAMP3 as a negative control. Luminescence values and the respective mean RLU value ± SEM observed after adding Nano-Glo Live Cell Reagent in each experiment are reported in the bar graph. A minimum of n = 8 experiments was performed for each experimental condition

Article Snippet: Non-specific blocking was done by incubation with 5% fat milk in PBS, 0.1% Tween 20 for 1 h. Blots were incubated overnight with 5% fat milk in PBS, 0.1% Tween 20, containing mouse monoclonal anti-STIM1 (CDN3H4 clone, Santa Cruz; 1:1,000 dilution; sc-66173) or mouse monoclonal anti-STIM1 (GOK BD, Alomone; 1:1,000 dilution; ACC-063) or mouse monoclonal anti-GAPDH antibody (6C5 clone, Abcam; 1:10,000 dilution).

Techniques: Clinical Proteomics, Membrane, Immunofluorescence, Labeling, Incubation, Ligation, Protein-Fragment Complementation Assay, Transfection, Positive Control, Negative Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A dual topology of STIM1 at the plasma membrane regulates calcium constitutive entry

doi: 10.1007/s00018-026-06141-0

Figure Lengend Snippet: STIM1 PM is implicated in the constitutive calcium entry measured in Panc-1-Wt cells. CCE was measured in single Panc-1-Wt cells loaded with Fura-2 by measuring the amplitude of fluorescence ratio variation following external Ca 2+ concentration changes (Panels A1, A3, B1, and B3) or quantified as the slope of Mn 2+ quenching of Fura-2 (Panels A2, A4, B2, and B4). CCE values are expressed in each condition as a percentage of the average value for each respective control. Histograms display single values of CCE along with the mean amplitude value ± SEM of the n observations. Representative recordings of CCE measurements are presented for the different conditions and experimental approaches. Panel A: CCE was evaluated in cells overexpressing STIM1 (OE STIM1; N > 7; Panels A1 and A2) or under-expressing STIM1 (siSTIM1; N > 6; Panels A3 and A4) and compared to values obtained in cells transfected with an empty vector (EV) or a non-targeted siRNA (siCtrl). Panel B: The effects of anti-STIM1 antibodies on CCE were evaluated using both experimental approaches. Cells were incubated for an hour with an anti-STIM1 antibody targeting the N-terminal (anti-STIM1 Nter1: N = 11, Panels B1 and B2) or the C-terminal (anti-STIM1 Cter3; N = 10, Panels B3 and B4) domains of STIM1. For each experiment, cells were also incubated in a set of experiments with a control isotype. Data are analyzed by non-parametric Mann–Whitney analysis, * P < 0.05, ** P < 0.01, and *** P < 0.005

Article Snippet: Non-specific blocking was done by incubation with 5% fat milk in PBS, 0.1% Tween 20 for 1 h. Blots were incubated overnight with 5% fat milk in PBS, 0.1% Tween 20, containing mouse monoclonal anti-STIM1 (CDN3H4 clone, Santa Cruz; 1:1,000 dilution; sc-66173) or mouse monoclonal anti-STIM1 (GOK BD, Alomone; 1:1,000 dilution; ACC-063) or mouse monoclonal anti-GAPDH antibody (6C5 clone, Abcam; 1:10,000 dilution).

Techniques: Fluorescence, Concentration Assay, Control, Expressing, Transfection, Plasmid Preparation, Incubation, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A dual topology of STIM1 at the plasma membrane regulates calcium constitutive entry

doi: 10.1007/s00018-026-06141-0

Figure Lengend Snippet: N-Glycosylation of STIM1 Regulates the Presence and Orientation of STIM1 PM in Panc-1 Wt Cells. Changes in STIM1 PM orientation following deglycosylation with a 12-h treatment with tunicamycin (5 µg/ml) were evaluated in Panc-1 Wt cells by flow cytometry, ELISA, and detection of HiBiT-tagged STIM1. Panels A-B: STIM1 mAb reactivity was assessed by flow cytometry ( n > 4) or ELISA ( n > 7) with non-permeabilized Panc-1-Wt cells using anti-STIM1 targeting the N-terminal (STIM1 Nter1: Panels A1 and B1) or targeting the C-terminal (STIM1 Cter3: Panels A2 and B2) domains of STIM1. Representative flow-cytometry overlays obtained following staining with anti-STIM1 antibodies are presented. Panel C: The amount of STIM1 PM at the plasma membrane was also evaluated in Panc-1 Wt cells expressing HiBiT-STIM1 (Panel C1) or STIM1-HiBiT (Panel C2) using the Promega Nano-Glo® HiBiT Detection System. Histograms represent the individual values of the mean Mean Fluorescence Intensity (MFI) or individual values of optical density (OD). In each histogram, the average ± SEM is also reported. Values are normalized to the average measured in control conditions and expressed as a percentage of this mean control value. Panel D: Representative Western blot of lysates from control and tunicamycin-treated cells showing the appearance of a band close to 75 kDa corresponding to the non-glycosylated form of STIM1. Panels E–F: CCE (Panel E) measured as the slope of the Mn 2+ quenching of Fura-2 fluorescence and SOCE (Panel F) were evaluated in cells left untreated or treated with tunicamycin. Representative recordings of CCE and SOCE are presented for the different conditions. For each parameter, individual values are presented as a percentage of the average values measured in non-treated cells. Data are analyzed by non-parametric Mann–Whitney analysis, * P < 0.05, ** P < 0.01, and *** P < 0.005

Article Snippet: Non-specific blocking was done by incubation with 5% fat milk in PBS, 0.1% Tween 20 for 1 h. Blots were incubated overnight with 5% fat milk in PBS, 0.1% Tween 20, containing mouse monoclonal anti-STIM1 (CDN3H4 clone, Santa Cruz; 1:1,000 dilution; sc-66173) or mouse monoclonal anti-STIM1 (GOK BD, Alomone; 1:1,000 dilution; ACC-063) or mouse monoclonal anti-GAPDH antibody (6C5 clone, Abcam; 1:10,000 dilution).

Techniques: Glycoproteomics, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Clinical Proteomics, Membrane, Expressing, Fluorescence, Control, Western Blot, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A dual topology of STIM1 at the plasma membrane regulates calcium constitutive entry

doi: 10.1007/s00018-026-06141-0

Figure Lengend Snippet: STIM1 glycosylation at positions N131 and N171 contributes to the orientation of STIM1 PM. Panel A: The amount of STIM1 PM at the plasma membrane is evaluated by ELISA in Panc-1 Wt cells transfected with expression vectors containing wild-type (WT) STIM1 (V5-STIM1-Flag), STIM1 mutated at the glycosylation sites (V5-STIM1-Flag N131/N171), or an empty vector. Cells were labeled with anti-STIM1 antibodies targeting the N-terminal (anti-STIM1 Nter1, anti-V5 Abs, Panel A1) or the C-terminal (anti-STIM1 Cter3, anti-Flag Abs, Panel A3) domains of STIM1. Histograms represent individual values of optical density (OD) and the mean value ± SEM of n observations ( n > 7) for each experimental condition. Values are normalized to the average OD value measured in cells transfected with the empty vector and expressed as a percentage of control. Panel B: The impact of glycosylation on STIM1 PM insertion in the plasma membrane was also evaluated in Panc-1 Wt cells using expression of HiBiT-tagged STIM1 constructs. Cells were transfected with HiBiT constructs containing wild-type STIM1 (HiBiT-STIM1 or STIM1-HiBiT) or STIM1 mutated at both glycosylation sites (HiBiT-STIM1 N131/N171Q or STIM1-HiBiT N131/171Q). Luminescence values are reported in bar graphs along with the mean value ± SEM of n observations ( n > 6). Values are normalized to the average luminescence value measured in cells expressing wild-type STIM1. Panel C: The role of STIM1 N-glycosylation on STIM1 PM multimerization was evaluated using NanoLuc® Binary Technology (NanoBiT). Cells were transfected with STIM1-LrgBiT and SmBiT-STIM1 constructs containing WT STIM1 or N131/N171Q mutated STIM1. Individual luminescence values and the respective mean RLU value ± SEM are reported in the bar graph. A minimum of n = 7 experiments was performed for each experimental condition. Data are expressed as the percentage of the average RLU value measured in cells expressing constructs with WT STIM1. Panel D: Representative Western blot of STIM1 detected with anti-STIM1 Nter1 expression in cells overexpressing V5-STIM1-Flag or V5-STIM1-Flag N131/171Q. Only the non-glycosylated form of STIM1 corresponding to the lower 85 kDa band is detected in cells expressing the glycosylation-mutated STIM1. Panels E–F: CCE evaluated by the slope of the Mn 2+ quenching of Fura-2 (Panel E) and SOCE (Panel F) are measured in cells overexpressing V5-STIM1-Flag or V5-STIM1-Flag N131/N171Q and compared to what was obtained in cells transfected with an empty vector (EV). Representative recordings of CCE and SOCE are presented for the different conditions. Values are expressed as a percentage of the average value obtained in cells transfected with an empty vector. Individual values and the mean value ± SEM of n observations ( n > 7) are presented in the different histograms. Data are analyzed by non-parametric Mann–Whitney analysis, ** P < 0.01 and *** P < 0.001

Article Snippet: Non-specific blocking was done by incubation with 5% fat milk in PBS, 0.1% Tween 20 for 1 h. Blots were incubated overnight with 5% fat milk in PBS, 0.1% Tween 20, containing mouse monoclonal anti-STIM1 (CDN3H4 clone, Santa Cruz; 1:1,000 dilution; sc-66173) or mouse monoclonal anti-STIM1 (GOK BD, Alomone; 1:1,000 dilution; ACC-063) or mouse monoclonal anti-GAPDH antibody (6C5 clone, Abcam; 1:10,000 dilution).

Techniques: Glycoproteomics, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Plasmid Preparation, Labeling, Control, Construct, Western Blot, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A dual topology of STIM1 at the plasma membrane regulates calcium constitutive entry

doi: 10.1007/s00018-026-06141-0

Figure Lengend Snippet: Insertion and orientation of STIM1 in the plasma membrane depends on a short C-terminal domain located close to the transmembrane domain. Panel A: The amount of STIM1 PM at the plasma membrane is evaluated by ELISA in non-permeabilized (Panels A1 and A2) or permeabilized (Panel A3) Panc-1 Wt cells transfected with expression vectors containing wild-type STIM1 (V5-STIM1-Flag), deleted STIM1 (V5-STIM1-Flag del 207–212), or an empty vector. Antibodies targeting the N-terminal (anti-STIM1 Nter1 and anti-V5 Abs, Panels A1 and A3) or targeting the C-terminal (anti-STIM1 Cter3 and anti-Flag Abs, Panels A2 and A3) domains of STIM1. Histograms represent individual values of normalized optical density (OD) and the mean ± SEM of n observations ( n > 7) for each experimental condition. Values were normalized to the average OD value obtained in cells transfected with the empty vector. Panel B: The amount of STIM1 PM at the plasma membrane is evaluated in Panc-1 Wt cell lines transfected with expression vectors containing wild-type V5-STIM1-Flag or STIM1 mutated at the pre-transmembrane sites (V5-STIM1-Flag del 207–212) by cytometry using antibodies targeting the N-terminal (STIM1 Nter1: GOK clone, Panel B1) or targeting the C-terminal (STIM1 Cter3: CDN3H4 clone, Panel B2). Histograms represent the mean Mean Fluorescence Intensity (MFI) ± SEM of STIM1 PM expressing cells ( n > 4). Panels C-D: CCE evaluated in cells overexpressing V5-STIM1-Flag or V5-STIM1-Flag del 207–212 using the Mn 2+ quench approach (Panel C) and SOCE recorded after store depletion and Ca 2+ addition in the extracellular medium (Panel D). Representative recordings of CCE and SOCE are presented for the different experimental conditions. Histograms present individual values of each parameter and the mean ± SEM of n observations ( n > 12) for these parameters. Data are expressed as the percentage of the mean value obtained for each parameter in cells transfected with an empty vector (EV). Data are analyzed by non-parametric Mann–Whitney analysis, ** P < 0.01 and *** P < 0.001

Article Snippet: Non-specific blocking was done by incubation with 5% fat milk in PBS, 0.1% Tween 20 for 1 h. Blots were incubated overnight with 5% fat milk in PBS, 0.1% Tween 20, containing mouse monoclonal anti-STIM1 (CDN3H4 clone, Santa Cruz; 1:1,000 dilution; sc-66173) or mouse monoclonal anti-STIM1 (GOK BD, Alomone; 1:1,000 dilution; ACC-063) or mouse monoclonal anti-GAPDH antibody (6C5 clone, Abcam; 1:10,000 dilution).

Techniques: Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Plasmid Preparation, Cytometry, Fluorescence, MANN-WHITNEY