mouse monoclonal anti stim1  (Alomone Labs)

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

Journal: Molecular Oncology

Article Title: Postbiotics of Lacticaseibacillus paracasei CECT 9610 and Lactiplantibacillus plantarum CECT 9608 attenuates store‐operated calcium entry and FAK phosphorylation in colorectal cancer cells

doi: 10.1002/1878-0261.13629

Figure Lengend Snippet: Expression of Orai and stromal interaction molecule (STIM) proteins and Store‐operated Ca 2+ entry (SOCE) is enhanced in the colorectal adenocarcinoma cell lines HT‐29 and Caco‐2. (A–E) NCM460, HT‐29 and Caco‐2 cells were lysed and the whole cell lysates were analyzed by western blotting using anti‐Orai1 (A), anti‐Orai2 (B), anti‐Orai3 (C), anti‐STIM1 (D) or anti‐STIM2 (E) antibody. Molecular masses indicated on the right were determined using molecular‐mass markers run in the same gel. Membranes were probed with anti‐β‐actin antibody for protein loading control. These results are representative of four separate experiments. Quantification of protein expression in NCM460 ( n = 4), HT‐29 ( n = 4) and Caco‐2 ( n = 4) cells normalized to the β‐actin expression is depicted in the bar graph. Data are represented as mean ± standard error of the mean (SEM) and were statistically analyzed using Kruskal–Wallis test combined with Dunn's post hoc test. ** P < 0.01 and **** P < 0.0001. (F) Representative Ca 2+ mobilization in response to 2 μ m thapsigargin (TG) measured using fura‐2 in NCM460 ( n = 3 [62 cells]), HT‐29 ( n = 3 [69 cells]) and Caco‐2 ( n = 3 [36 cells]) cells. Cells were superfused with a Ca 2+ ‐free Hepes Buffer Saline (HBS) (100 μ m ethylene glycol‐bis(2‐aminoethylether)‐ N , N , N ′, N ′‐tetraacetic acid (EGTA) added) and stimulated with 2 μ m TG, followed by re‐addition of CaCl 2 (1.8 m m ) to estimate Ca 2+ influx. (G, H) Quantification of TG‐evoked Ca 2+ release from the intracellular stores and entry in NCM460 ( n = 3 [62 cells]), HT‐29 ( n = 3 [69 cells]) and Caco‐2 ( n = 3 [36 cells]) cells is shown in the scatter plots. Data in bar graphs are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test combined with Dunn's post hoc test. **** P < 0.0001. a.u., arbitrary units; AUC, area under the curve.

Article Snippet: Mouse monoclonal Anti‐GOK/Stim1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Expressing, Western Blot, Saline

Journal: Molecular Oncology

Article Title: Postbiotics of Lacticaseibacillus paracasei CECT 9610 and Lactiplantibacillus plantarum CECT 9608 attenuates store‐operated calcium entry and FAK phosphorylation in colorectal cancer cells

doi: 10.1002/1878-0261.13629

Figure Lengend Snippet: Effect of exposure to heat‐killed Lacticaseibacillus paracasei or Lactiplantibacillus plantarum on store‐operated Ca 2+ entry (SOCE) in NCM460 cells. NCM460 cells were exposed for 24 and 48 h to heat‐killed Lacticaseibacillus paracasei or Lactiplantibacillus plantarum (10 8 colony forming units (CFU)·mL −1 ). (A, B) Representative Ca 2+ mobilization in response to 2 μ m thapsigargin (TG) measured using fura‐2 in NCM460 cells. Cells were superfused with a Ca 2+ ‐free Hepes Buffer Saline (HBS) (100 μ m ethylene glycol‐bis(2‐aminoethylether)‐ N , N , N ′, N ′‐tetraacetic acid (EGTA) added) and stimulated with 2 μ m TG, followed by re‐addition of CaCl 2 (1.8 m m ) to estimate Ca 2+ influx. Quantification of TG‐evoked Ca 2+ release from the intracellular stores (C) and entry (D) in non‐exposed ( n = 4 [150 cells]), 24 h Lacticaseibacillus paracasei exposed ( n = 4 [96 cells]), 48 h Lacticaseibacillus paracasei exposed ( n = 4 [85 cells]), 24 h Lactiplantibacillus plantarum exposed ( n = 4 [83 cells]), 48 h Lactiplantibacillus plantarum exposed ( n = 4 [87 cells]) NCM460 cells is shown in the scatter plots. Data are represented as mean ± standard error of the mean (SEM) and were statistically analyzed using Kruskal–Wallis test (no significant differences were found). (E–H) NCM460 cells were exposed for 24 and 48 h to heat‐killed Lacticaseibacillus paracasei or Lactiplantibacillus plantarum (10 8 CFU·mL −1 ), or left untreated, and lysed. The whole cell lysates were analyzed by western blotting using anti‐Orai1 (E), anti‐Orai3 (F), anti‐STIM1 (G) or anti‐STIM2 (H). Molecular masses indicated on the right were determined using molecular‐mass markers run in the same gel. Membranes were probed with anti‐β‐actin antibody for protein loading control. These results are representative of four separate experiments. Quantification of Orai and STIM protein expression normalized to the β‐actin expression is depicted in the bar graphs. Data are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test. a.u., arbitrary units; AUC, area under the curve.

Article Snippet: Mouse monoclonal Anti‐GOK/Stim1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Saline, Western Blot, Expressing

Journal: Molecular Oncology

Article Title: Postbiotics of Lacticaseibacillus paracasei CECT 9610 and Lactiplantibacillus plantarum CECT 9608 attenuates store‐operated calcium entry and FAK phosphorylation in colorectal cancer cells

doi: 10.1002/1878-0261.13629

Figure Lengend Snippet: Effect of HT‐29 cell exposure to heat‐killed Lacticaseibacillus paracasei or Lactiplantibacillus plantarum on store‐operated Ca 2+ entry (SOCE) and Orai and STIM expression. (A–D) HT‐29 cells were exposed for 24 and 48 h to heat‐killed Lacticaseibacillus paracasei or Lactiplantibacillus plantarum (10 8 colony forming units (CFU)·mL −1 ), or left untreated. (A, B) Representative Ca 2+ mobilization in response to 2 μ m thapsigargin (TG) measured using fura‐2 in HT‐29 cells. Cells were superfused with a Ca 2+ ‐free Hepes Buffer Saline (HBS) (100 μ m ethylene glycol‐bis(2‐aminoethylether)‐ N , N , N ′, N ′‐tetraacetic acid (EGTA) added) and stimulated with 2 μ m TG, followed by re‐addition of CaCl 2 (1.8 m m ) to estimate Ca 2+ influx. Quantification of TG‐evoked Ca 2+ release (C) and entry (D) in non‐exposed ( n = 4 [120 cells]), 24 h Lacticaseibacillus paracasei exposed ( n = 4 [105 cells]), 48 h Lacticaseibacillus paracasei exposed ( n = 4 [89 cells]), 24 h Lactiplantibacillus plantarum exposed ( n = 4 [87 cells]), 48 h Lactiplantibacillus plantarum exposed ( n = 4 [84 cells]) HT‐29 cells is shown in the scatter plots. Data are represented as mean ± standard error of the mean (SEM) and were statistically analyzed using Kruskal–Wallis test combined with Dunn's post hoc test. *** P < 0.001 and **** P < 0.0001. (E‐H) HT‐29 cells were exposed for 24 and 48 h to heat‐killed Lacticaseibacillus paracasei or Lactiplantibacillus plantarum (10 8 CFU·mL −1 ), or left untreated, and lysed. The whole cell lysates were analyzed by western blotting using anti‐Orai1 (E), anti‐Orai3 (F), anti‐STIM1 (G) or anti‐STIM2 (H). Molecular masses indicated on the right were determined using molecular‐mass markers run in the same gel. Membranes were probed with anti‐β‐actin antibody for protein loading control. These results are representative of four separate experiments. Quantification of Orai and STIM protein expression normalized to the β‐actin expression is depicted in the bar graphs. Data are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test combined with Dunn's post hoc test. ** P < 0.01, *** P < 0.001 and **** P < 0.0001. a.u., arbitrary units; AUC, area under the curve.

Article Snippet: Mouse monoclonal Anti‐GOK/Stim1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Expressing, Saline, Western Blot

Journal: Molecular Oncology

Article Title: Postbiotics of Lacticaseibacillus paracasei CECT 9610 and Lactiplantibacillus plantarum CECT 9608 attenuates store‐operated calcium entry and FAK phosphorylation in colorectal cancer cells

doi: 10.1002/1878-0261.13629

Figure Lengend Snippet: Effect of exposure to heat‐killed Lacticaseibacillus paracasei or Lactiplantibacillus plantarum on store‐operated Ca 2+ entry (SOCE) and Orai and STIM expression in Caco‐2 cells. (A–D) Caco‐2 cells were exposed for 24 and 48 h to heat‐killed Lacticaseibacillus paracasei or Lactiplantibacillus plantarum (10 8 colony forming units (CFU)·mL −1 ), or left untreated. (A, B) Representative Ca 2+ mobilization in response to 2 μ m thapsigargin (TG) measured using fura‐2 in Caco‐2. Cells were superfused with a Ca 2+ ‐free Hepes Buffer Saline (HBS) (100 μ m ethylene glycol‐bis(2‐aminoethylether)‐ N , N , N ′, N ′‐tetraacetic acid (EGTA) added) and stimulated with 2 μ m TG, followed by re‐addition of CaCl 2 (1.8 m m ) to estimate Ca 2+ influx. Quantification of TG‐evoked Ca 2+ release (C) and entry (D) in non‐exposed ( n = 3 [44 cells]), 24 h Lacticaseibacillus paracasei exposed ( n = 3 [39 cells]), 48 h Lacticaseibacillus paracasei exposed ( n = 3 [30 cells]), 24 h Lactiplantibacillus plantarum exposed ( n = 3 [31 cells]), 48 h Lactiplantibacillus plantarum exposed ( n = 3 [34 cells]) Caco‐2 cells is shown in the scatter plots. Data are represented as mean ± standard error of the mean (SEM) and were statistically analyzed using Kruskal–Wallis test combined with Dunn's post hoc test. ** P < 0.01 and **** P < 0.0001. (E–H) Caco‐2 cells were exposed for 24 and 48 h to heat‐killed Lacticaseibacillus paracasei or Lactiplantibacillus plantarum (10 8 CFU·mL −1 ), or left untreated, and lysed. The whole cell lysate was analyzed by western blotting using anti‐Orai1 (E), anti‐Orai3 (F), anti‐STIM1 (G) or anti‐STIM2 (H). Molecular masses indicated on the right were determined using molecular‐mass markers run in the same gel. Membranes were probed with anti‐β‐actin antibody for protein loading control. These results are representative of four separate experiments. Quantification of Orai and STIM protein expression normalized to the β‐actin expression is depicted in the bar graphs. Data are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test combined with Dunn's post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. a.u., arbitrary units; AUC, area under the curve.

Article Snippet: Mouse monoclonal Anti‐GOK/Stim1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Expressing, Saline, Western Blot

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: ( A ) Fura-2-loaded wild type HEK-293 cells (WT) and STIM1,2-DKO HEK-293 cells (DKO) were suspended in a Ca 2+ -free (100 µM EGTA) HBS and then stimulated with 2 µM TG followed by reintroduction of external Ca 2+ (final concentration 1.8 mM) to initiate Ca 2+ entry. ( B and C ) DKO HEK-239 cells were co-transfected with either CMV-driven Orai1α-eGFP (O1α; B ) or Orai1β-eGFP (O1β; C ) and either STIM1-YFP (S1), STIM2-YFP (S2) or both (S1+S2) plasmids, as described. Forty-eight hours later cells were loaded with fura-2 and suspended in a Ca 2+ -free HBS (100 µM EGTA added) and then stimulated with TG (2 µM) followed by the addition of extracellular Ca 2+ (final concentration 1.8 mM) to initiate Ca 2+ influx. ( D and E ) quantification of TG-evoked Ca 2+ release ( D ) and entry ( E ) determined as described in materials and methods. Bar graphs are represented as mean ± SEM and expressed as fold change over control (WT HEK-293 cells). From left to right, n[=[224, 112, 50, 46, 37, 41, 41 and 23; n values correspond to individual cells. Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunńs test). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 as compared to WT HEK-293 cells. $$ P < 0.01 and $$$$ P < 0.0001 as compared to DKO HEK-293 cells. φ P < 0.05 as compared to DKO cells transfected with STIM1 expressing plasmid. ( F ) WT HEK-293 cells (lane 1) and DKO cells either transfected with empty vector or with CMV-driven Orai1α or Orai1β in combination with either STIM1, STIM2 or both plasmids (lanes 2-8) were lysed and then subjected to 10% SDS-PAGE and Western blotting with the anti-STIM2, anti-STIM1 or anti-Orai1 (C-terminal) antibody, as described in material and methods. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments.

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Concentration Assay, Transfection, Expressing, Plasmid Preparation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: ( A - F ) Representative Ca 2+ mobilization in response to 10[µM CCh measured using fura-2 in wild type HEK-293 cells (WT), STIM1,2-DKO HEK-293 cells (DKO) and DKO cells co-transfected with either CMV-driven Orai1α-eGFP (O1α) or Orai1β-eGFP (O1β) and either STIM1-YFP (S1), STIM2-YFP (S2) or both (S1+S2) plasmids, as described. Cells were superfused with HBS containing 2[mM Ca 2+ and stimulated with 10[µM CCh at 1[min (indicated by arrow). Representative traces from five cells/condition were chosen to represent the datasets. ( G ) Quantification of Ca 2+ mobilization for all the conditions from A to F estimated in all the cells. (for G , from left to right, n =110, 141, 67, 34, 89 and 70; n-values correspond to individual cells). Scatter plots are represented as mean[±[SEM and expressed as fold change over control (WT HEK-293 cells). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunńs test). **** P < 0.0001 as compared to WT HEK-293 cells. $$ P < 0.01, $$$ P < 0.001 and $$$$ P < 0.0001 as compared to DKO cells. φ P < 0.05 as compared to DKO cells transfected with STIM1 expressing plasmid.

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: ( A ) Fura-2-loaded wild type HEK-293 cells (WT) and STIM1,2-DKO HEK-293 cells (DKO) were suspended in a Ca 2+ -free HBS (100 µM EGTA) and then stimulated with 2 µM TG followed by reintroduction of external Ca 2+ (final concentration 1.8 mM) to initiate Ca 2+ entry. ( B and C ) DKO cells were co-transfected with either shOrai1 and TK-driven Orai1α-eGFP (O1α; B ) or Orai1β-eGFP (O1β; C ) and either STIM1-GFP (S1) or STIM2-GFP (S2) plasmids. Forty-eight hours later cells were loaded with fura-2 and suspended in a Ca 2+ -free HBS (100 µM EGTA added) and then stimulated with TG (2 µM) followed by the addition of extracellular Ca 2+ (final concentration 1.8 mM) to initiate Ca 2+ influx. ( D and E ) quantification of TG-evoked Ca 2+ release ( D ) and entry ( E ) determined as described in materials and methods. Bar graphs are represented as mean ± SEM and expressed as fold change over control (WT HEK-293 cells). From left to right, n[=[203, 133, 105, 82, 70 and 109; n values correspond to individual cells. Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunńs test). ** P < 0.01 and **** P < 0.0001 as compared to WT HEK-293 cells. $$$ P < 0.001 and $$$$ P < 0.0001 as compared to DKO cells. φφφ P < 0.001 and φφφφ P < 0.0001 as compared to DKO cells transfected with STIM1 expressing plasmid. ( F ) WT HEK-293 cells (lane 1) and DKO cells either transfected with empty vector or with TK-driven Orai1α or Orai1β in combination with either STIM1, STIM2 or both plasmids (lanes 2-8), as described, were lysed and then subjected to 10% SDS-PAGE and Western blotting with the anti-STIM2, anti-STIM1 or anti-Orai1 (C-terminal) antibody, as described in material and methods. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments.

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Concentration Assay, Transfection, Expressing, Plasmid Preparation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: ( A-F ) Representative Ca 2+ mobilization in response to 10[µM CCh measured using fura-2 in wild type HEK-293 cells (WT), STIM1,2-DKO HEK-293 cells (DKO) and DKO cells co-transfected with either shOrai1 and TK-driven Orai1α-eGFP (O1α) or Orai1β-eGFP (O1β) and either STIM1-GFP (S1) or STIM2-GFP (S2) plasmids, as described. Cells were superfused with HBS containing 2[mM Ca 2+ and stimulated with 10[µM CCh at 1[min (indicated by arrow). Representative traces from five cells/condition were chosen to represent the datasets. ( G ) Quantification of Ca 2+ mobilization for all the conditions from A to F estimated in all the cells. (for G , from left to right, n =187, 286, 91, 61, 110 and 126; n-values correspond to individual cells). ( H-J ) Quantification of the percentage of oscillating cells ( H ), non-oscillating cells ( I ) and total oscillations/cell in 20 min ( J ) for data presented in A – F (for H to J , from left to right, n[=[8, 8, 6, 5, 5 and 6; n values correspond to independent experiments). Scatter plots are represented as mean[±[SEM and expressed as fold change over control (WT HEK-293 cells). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunńs test). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 as compared to WT HEK-293 cells. $ P < 0.05, and $$$$ P < 0.0001 as compared to DKO cells. φφφφ P < 0.0001 as compared to DKO cells transfected with STIM1 expressing plasmid.

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: ( A-D ) Resting (control) and stimulated (2 µM TG for 1 min) WT HEK293 cells co-expressing Orai1α-eGFP ( A and C ) or Orai1β-eGFP ( B and D ) with STIM1-mCherry ( A and B ) or STIM2-mCherry ( C and D ), their overlay ( A-D , Overlay) and calculated FRET values ( A-D , FRET) are presented. Cells were superfused with a medium containing 1.8 mM Ca 2+ . The FRET values depicted in E were calculated from the averages of whole cell areas determined from the respective number of cells showing significantly increased FRET values. Values are mean ± S.E. of twenty independent experiments. Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunńs test). * P < 0.05, **** P < 0.0001, $$$$ P < 0.0001 as compared to their respective controls (untreated cells). The scale bar in the box represents 10 µm.

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Expressing

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: STIM1,2-DKO HEK-293 cells were left untreated ( A , lanes 1 and 2) or co-transfected with CMV-driven Orai1α-eGFP ( A , lanes 3-8) or Orai1β-eGFP ( B , lanes 3-8) in combination with either STIM1-YFP, STIM2-YFP or both plasmids. Forty-eight hours later cells were then treated in the absence or presence of 2 µM TG for 1 min and lysed. Cell lysates were then subjected to 10% SDS-PAGE and Western blotting with the anti-STIM2, anti-STIM1 or anti-Orai1 (C-terminal) antibody, as described in material and methods. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four to five separate experiments. ( C-D ) Quantification of the interaction of STIM1 ( C ) or STIM2 ( D ) interaction with Orai1α or Orai1β. Data were statistically analyzed using Mann–Whitney U test. * P < 0.05 and ** P < 0.01 as compared to their respective controls (untreated cells).

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Transfection, SDS Page, Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: STIM1,2-DKO HEK-293 cells were left untreated (panels A and B , lanes 1 and 2) or co-transfected with CMV-driven Orai1α-eGFP ( A , lanes 3-10) or Orai1β-eGFP ( B , lanes 3-10) alone or in combination with either STIM1-YFP, STIM2-YFP or both plasmids, as indicated. Forty-eight hours later cells were then treated in the absence (Ctrl) or presence of 2 µM TG for 1 min and lysed. Samples were then mixed with biotinylation buffer containing EZ-Link sulfo-NHS-LC-biotin, and cell surface proteins were labeled by biotinylation. Labeled proteins were pulled down with streptavidin-coated agarose beads. The pellet (containing the plasma membrane fraction) and the supernatant were analyzed by SDS-PAGE and Western blot analysis using anti-Orai1 C-terminal antibody, as indicated. Membranes were probed with anti-PMCA antibody. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. These results are representative of 6 separate experiments. ( C-D ) Quantification of Orai1 plasma membrane expression under the different experimental conditions normalized to the PMCA expression is depicted in the bar graph. Data are represented as mean[±[SEM and expressed as fold increase (experimental/control). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn’s test). $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 and $$$$ P < 0.0001 as compared to their respective control (untreated cells). **** P < 0.0001 as compared to the Orai1 expression in the absence of STIM proteins. ( E ) HEK-293 cells were transfected with CMV-driven Orai1α-eGFP or Orai1β-eGFP alone or in combination with CMV-driven STIM1-mCherry or STIM2-mCherry. Forty-eight hours later GFP fluorescence was detected using an LSM900 confocal microscope. The images show representative confocal images of Orai1α-eGFP or Orai1β-eGFP. The scale bar represents 10 μm. Scatter plots represent GFP intensity at the different experimental conditions. Data are presented as mean ± SEM and are statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunńs test). *** P < 0.001 and **** P < 0.0001 as compared to HEK-293 cells transfected with Orai1α-eGFP or Orai1β-eGFP alone.

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Transfection, Labeling, Membrane, SDS Page, Western Blot, Expressing, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: STIM1,2-DKO HEK-293 cells were co-transfected with CMV-driven Orai1α ( A ) or Orai1β ( B ) in combination with either STIM1-YFP, STIM2-YFP or the STIM1-D76A and STIM2-D80A-YFP mutants, with a relative Orai1:STIM expression ratio of 1:1 or 1:2, as indicated. Forty-eight hours later cells were lysed, and cell lysates were analyzed by SDS-PAGE and Western blot analysis using anti-Orai1 C-terminal antibody. Membranes were probed with anti-STIM1, anti-STIM2 or anti β-actin antibody ( A-C ). Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. These results are representative of 6 separate experiments. Bar graphs represent the quantification of Orai1α, Orai1β, STIM1 and STIM2 expression under the different experimental conditions normalized to the β-actin expression. Data are represented as mean[±[SEM and expressed as fold increase (experimental/control). ( A-B ) Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn’s test). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 as compared to the expression of Orai1α or Orai1β in the absence of STIM. $ P < 0.05 and $$ P < 0.01 as compared to the Orai1 expression at Orai1:STIM expression ratio of 1:1. # P < 0.05 as compared to the Orai1 expression in the presence of WT STIM proteins. ( C ) Data were statistically analyzed using Mann-Whitney U test. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 as compared to the expression of STIM1 or STIM2 at Orai1:STIM relative expression ratio of 1:1. ( D ). STIM1,2-DKO HEK-293 cells were co-transfected with CMV-driven Orai1α in combination with either STIM1 D76A or STIM2-D80A mutant, with empty vector (Control). Forty-eight hours later cells were loaded with fura-2 and suspended in a Ca 2+ -free HBS (100 µM EGTA added). Ca 2+ was added to the extracellular medium at a final concentration of 1.8 mM to initiate Ca 2+ influx.

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Transfection, Expressing, SDS Page, Western Blot, MANN-WHITNEY, Mutagenesis, Plasmid Preparation, Concentration Assay

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: (A) HEK-293 cells were transfected with empty vector (Ctrl), STIM1-YFP or STIM2-YFP expression plasmids or with shSTIM1 or esiSTIM2, as indicated. Forty-eight hours later cells lysed. Cell lysates were treated with PNGaseF and then subjected to 10% SDS-PAGE and Western blot analysis using anti-STIM1, anti-STIM2 or anti-Orai1 C-terminal antibody. Membranes were reprobed with anti β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. These results are representative of 6 separate experiments. ( B-E ) Quantification of Orai1α, Orai1β, STIM1 and STIM2 expression under the different experimental conditions normalized to the β-actin expression. Data are represented as mean[±[SEM and expressed as fold change (experimental/control). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn’s test). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 as compared to the protein expression in WT cells.

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: STIM1,2-DKO HEK-293 cells were co-transfected with CMV-driven Orai1α-eGFP ( A, C, E ) or Orai1β-eGFP ( B, D, F ) in combination with either STIM1-YFP, STIM2-YFP or both plasmids, as indicated. Forty-eight hours later cells were either treated with cycloheximide (CHX; 10 µM) or the vehicle for 7 h and lysed ( A - B ) or were treated with cycloheximide (10 µM) alone or in combination with 10 µM MG132 for 7 h and lysed ( C - D ) or were treated with cycloheximide (10 µM) alone or in combination with 1 µM bafilomycin A1 (BafA1) for 7 h and lysed ( E - F ). Cell lysates were analyzed by SDS-PAGE and Western blot analysis using anti-STIM1, anti-STIM2 or anti-Orai1 C-terminal antibody. Membranes were reprobed with anti β-actin antibody. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. These results are representative of 6 separate experiments. Bar graphs show quantification of Orai1α and Orai1β expression under the different experimental conditions normalized to the β-actin expression. Data are represented as mean[±[SEM and expressed as fold change (experimental/control). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn’s test). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 as compared to the Orai expression in the absence of STIM proteins.

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Transfection, SDS Page, Western Blot, Expressing

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: ( A ) STIM1,2-DKO HEK-293 cells were co-transfected with CMV-driven Orai1α-eGFP or a dominant negative Orai1α mutant (Orai1αdn), in combination with either STIM1-YFP, STIM2-YFP or the STIM1 OASF fragment, as indicated. Forty-eight hours later cells were lysed and cell lysates were analyzed by SDS-PAGE and Western blot analysis using anti-STIM1, anti-STIM2 or anti-Orai1 C-terminal antibody. Membranes were reprobed with anti β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. These results are representative of 6 separate experiments. Bar graphs show quantification of Orai1α and Orai1αdn expression under the different experimental conditions normalized to the β-actin expression. Data are represented as mean[±[SEM and expressed as fold change (experimental/control). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn’s test). ** P < 0.01 and **** P < 0.0001 as compared to the expression of Orai1α or Orai1αdn in the absence of STIM proteins and fragments. (B) STIM1,2-DKO HEK-293 cells were co-transfected with CMV-driven Orai1α or a dominant negative Orai1α mutant (Orai1αdn), in combination with STIM1, as indicated. Forty-eight hours later cells were loaded with fura-2 and suspended in a Ca 2+ -free HBS (100 µM EGTA added) and then stimulated with TG (2 µM) followed by the addition of extracellular Ca 2+ (final concentration 1.8 mM) to initiate Ca 2+ influx. Bar graphs represent quantification of TG-evoked Ca 2+ release and entry determined as described in materials and methods. Data are presented as mean ± SEM and expressed as fold change over control (cells expressing Orai1α and STIM1). Data were statistically analyzed using Mann-Whitney U test. **** P < 0.0001 as compared to cells expressing Orai1α and STIM1. ( C ) STIM1,2-DKO HEK-293 cells were co-transfected with CMV-driven STIM1 OASF fragment. Forty-eight hours later GFP fluorescence was detected using an LSM900 confocal microscope. The images show representative confocal images of the STIM1 OASF region. The scale bar represents 100 μm. ( D ) STIM1,2-DKO HEK-293 cells were co-transfected with CMV-driven Orai1α or a dominant negative Orai1α mutant (Orai1αdn), in combination with STIM1 or the STIM1 OASF fragment, as indicated. Forty-eight hours later cells were loaded with fura-2 and suspended in a Ca 2+ -free HBS (100 µM EGTA added). Ca 2+ was added to the extracellular medium at a final concentration of 1.8 mM to initiate Ca 2+ influx.

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Transfection, Dominant Negative Mutation, Mutagenesis, SDS Page, Western Blot, Expressing, Concentration Assay, MANN-WHITNEY, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Feedback modulation of Orai1α and Orai1β protein content mediated by STIM proteins

doi: 10.1101/2024.03.05.583469

Figure Lengend Snippet: STIM1,2-DKO HEK-293 cells were co-transfected with CMV-driven STIM1-YFP ( A ) or STIM2-YFP ( B ) in combination with Orai1α-eGFP or Orai1β-eGFP, with a relative Orai1:STIM expression ratio of 1:2. Forty-eight hours later cells were lysed and cell lysates were analyzed by SDS-PAGE and Western blot analysis using anti-STIM1 or anti-STIM2 antibody. Membranes were probed with anti-Orai1 C-terminal antibody or anti β-actin antibody. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. These results are representative of 6 separate experiments. Bar graphs represent the quantification of STIM1 and STIM2 expression under the different experimental conditions normalized to the β-actin expression. Data are represented as mean[±[SEM and expressed as fold increase (experimental/control). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn’s test). **** P < 0.0001 as compared to the STIM1 expression in the absence of Orai1 variants.

Article Snippet: Mouse monoclonal Anti-GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Transfection, Expressing, SDS Page, Western Blot