Journal: Cells

Article Title: STIM Protein-NMDA2 Receptor Interaction Decreases NMDA-Dependent Calcium Levels in Cortical Neurons

doi: 10.3390/cells9010160

Figure Lengend Snippet: shSTIM1 and shSTIM2 increase NMDA-induced Ca 2+ responses. ( a ) Western blot analysis of STIM1 and STIM2 protein levels using anti-STIM1 and anti-STIM2 antibodies in cortical neurons transduced with lentiviruses expressing different shRNA sequences directed against RNA for STIM1 (A1, C1, or D1), for STIM2 (A2, C2, or D2) or the control sequence shRNA (sc1, sc2). GAPDH served as reference. ( b ) Results of quantitative WB analysis of cell lysates obtained from neurons transduced as in ( a ). Each column shows the mean ± SEM of three independent transductions. Statistical analysis performed by ANOVA, followed by Tukey’s Multiple Comparison Test. NT, non-transduced control, ** p < 0.01; *** p < 0.001. ( c – f ) NMDAR agonists-induced [Ca 2+ ] i responses increased when expression of STIM1 ( c , d ) and STIM2 ( e , f ) is silenced by shA and shC compared to neurons transduced with shsc control plasmid. F 340 /F 380 values just before adding NMDAR agonists normalized to the same values (1). Data represent m number of analyzed cells in n independent experiments that were conducted on three different primary cultures (NMDA_sc1, m = 89, n = 5), (NMDA_A1, m = 98, n = 5), (NMDA_C1, m = 96, n = 7), (NMDA_sc2, m = 104, n = 6), (NMDA_A2, m = 60, n = 7), and (NMDA_C2, m = 92, n = 9). ( d , f ) Summary of graphs ( c , e ) shown as an area under the curve (AUC). *** p < 0.001 significantly different compared with NMDA_sc (ANOVA followed by Tukey’s Multiple Comparison Test).

Article Snippet: The precipitated proteins were then washed three times with repeated centrifugation, eluted in 50 µL of 2× Laemmli Buffer, and subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) and western blot analysis with the indicated primary antibodies rabbit STIM1 (1:200, ProteinTech Group), rabbit STIM2 (1:100, Alomone Labs), rabbit NR2A (1:200, Merck Millipore) and rabbit NR2B (1:300, ProteinTech Group) at 4 °C overnight and then with the appropriate horseradish peroxidase-conjugated secondary antibody IgG (1:5000, Sigma) diluted in blocking solution (TBST: 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20 plus 5% dry non-fat milk).

Techniques: Western Blot, Transduction, Expressing, shRNA, Sequencing, Plasmid Preparation

Journal: Cells

Article Title: STIM Protein-NMDA2 Receptor Interaction Decreases NMDA-Dependent Calcium Levels in Cortical Neurons

doi: 10.3390/cells9010160

Figure Lengend Snippet: Neurons with overexpressed STIM1 and STIM2 exhibit lower NMDA-induced [Ca 2+ ] i . ( a ) Analysis of NMDA and glycine-induced [Ca 2+ ] i in the presence of nimodipine and CNQX based on ratiometric measurements with Fura2-AM in neurons overexpressing YFP-STIM1, YFP-STIM2, or YFP. F 340 /F 380 values immediately prior to adding the NMDAR agonist were normalized to the same values (1). Data represent n independent experiments that were conducted on h different primary cultures corresponding to 71 (YFP, n = 11, h = 4), 104 (YFP-STIM1, n = 16, h = 6) and 81 (YFP-STIM2, n = 16, h = 6) analyzed cells. ( b ) Summary of data from ( a ) shown as an AUC. *** p < 0.001 significantly different compared with YFP (ANOVA followed by Tukey’s Multiple Comparison Test). ( c ) Analysis of [Ca 2+ ] i induced by NMDA and glycine in the presence of nimodipine and CNQX based on ratiometric measurements with Fura2-AM in neurons from Tg(STIM1)Ibd (Tg) or control (wild type, WT) mice. F 340 /F 380 values immediately prior to adding the NMDAR agonist were normalized to the same values (1). The data represent n independent experiments that were conducted on four different primary cultures, corresponding to 196 (NMDA_WT, n = 20) and 183 (NMDA_STIM1_Tg, n = 17) analyzed cells. ( d ) Summary of data from ( c ) shown as AUC. * p < 0.05 significantly different compared with NMDA_WT (Mann–Whitney U test).

Article Snippet: The precipitated proteins were then washed three times with repeated centrifugation, eluted in 50 µL of 2× Laemmli Buffer, and subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) and western blot analysis with the indicated primary antibodies rabbit STIM1 (1:200, ProteinTech Group), rabbit STIM2 (1:100, Alomone Labs), rabbit NR2A (1:200, Merck Millipore) and rabbit NR2B (1:300, ProteinTech Group) at 4 °C overnight and then with the appropriate horseradish peroxidase-conjugated secondary antibody IgG (1:5000, Sigma) diluted in blocking solution (TBST: 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20 plus 5% dry non-fat milk).

Techniques: MANN-WHITNEY

Journal: Cells

Article Title: STIM Protein-NMDA2 Receptor Interaction Decreases NMDA-Dependent Calcium Levels in Cortical Neurons

doi: 10.3390/cells9010160

Figure Lengend Snippet: The interaction between endogenous STIM1/STIM2 and NR2A/NR2B occurs in situ. ( a ) Proximity ligation assay between STIM1 and NR2B, STIM1 and NR2A, STIM2 and NR2B, and STIM2 and NR2A before and after store depletion by TG/EGTA observed by fluorescent microscopy. Neurons were also counter-stained with the nuclear marker Hoechst dye (blue). The PLA signal, recognized as a fluorescent green dot, shows the close proximity of STIM and NR2 antigens. ( b ) No signals were observed when one of the primary antibodies was omitted (either anti-NR2A or NR2B or STIM1 or STIM2) that demonstrates the specificity of the detection assay and used antibodies. Scale bar, 10 µm for each panel. ( c ) Quantification of the complexes detected by PLA. Bars represent averages from 15–30 ( n ) images taken in three independent experiments, corresponding to 42–64 cells ± SEM. The quantification of PLA signals was performed using ImageJ software to analyse neurons. * p < 0.05; ** p < 0.01; ns, not significant compared with the control (Mann–Whitney U test). ( d ) The picture shows the higher magnification of one neuron from ( a ) to better visualize the PLA signals and their localization in the cell.

Article Snippet: The precipitated proteins were then washed three times with repeated centrifugation, eluted in 50 µL of 2× Laemmli Buffer, and subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) and western blot analysis with the indicated primary antibodies rabbit STIM1 (1:200, ProteinTech Group), rabbit STIM2 (1:100, Alomone Labs), rabbit NR2A (1:200, Merck Millipore) and rabbit NR2B (1:300, ProteinTech Group) at 4 °C overnight and then with the appropriate horseradish peroxidase-conjugated secondary antibody IgG (1:5000, Sigma) diluted in blocking solution (TBST: 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20 plus 5% dry non-fat milk).

Techniques: In Situ, Proximity Ligation Assay, Microscopy, Staining, Marker, Detection Assay, Software, MANN-WHITNEY

Journal: Cells

Article Title: STIM Protein-NMDA2 Receptor Interaction Decreases NMDA-Dependent Calcium Levels in Cortical Neurons

doi: 10.3390/cells9010160

Figure Lengend Snippet: Endogenous STIMs co-localize with NMDAR2. ( a ) Representative confocal images of Ca 2+ or TG/EGTA-treated neurons fixed and stained with anti-NR2A or NR2B antibody (shown in green, 1st panels), with anti-STIM1 or anti-STIM2 (shown in red, 2nd panels) and with anti-MAP2 to identify neurons for analysis (not shown for clarity). (Blue) Nuclei were stained with Hoechst. The 3rd columns show the merged images of green, red and blue channels. Co-localization of NR subunits with STIM is shown in the 4th columns (white). All images are taken from a single slice from the middle of the cell. Scale bar, 5 µm for each panel. ( b , c ) Results of co-localization analysis of NRs with STIMs in soma of neurons treated as in ( a ). Bar graph depicting the quantification of the average value of STIM with NR co-localization coefficient according to Manders ( b ) or the difference between this coefficient value 10 min after TG treatment and before store depletion (in the presence of 2 mM Ca 2+ ) ( c ). * p < 0.05; ** p < 0.01; ns , not significant compared with the control (Mann-Whitney U test). Bars represent the average of cultures from three animals; 35–55 cells per each condition (Ca 2+ or TG/EGTA).

Article Snippet: The precipitated proteins were then washed three times with repeated centrifugation, eluted in 50 µL of 2× Laemmli Buffer, and subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) and western blot analysis with the indicated primary antibodies rabbit STIM1 (1:200, ProteinTech Group), rabbit STIM2 (1:100, Alomone Labs), rabbit NR2A (1:200, Merck Millipore) and rabbit NR2B (1:300, ProteinTech Group) at 4 °C overnight and then with the appropriate horseradish peroxidase-conjugated secondary antibody IgG (1:5000, Sigma) diluted in blocking solution (TBST: 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20 plus 5% dry non-fat milk).

Techniques: Staining, MANN-WHITNEY

Journal: Cells

Article Title: STIM Protein-NMDA2 Receptor Interaction Decreases NMDA-Dependent Calcium Levels in Cortical Neurons

doi: 10.3390/cells9010160

Figure Lengend Snippet: Endogenous STIMs co-immunoprecipitate with NMDAR2. ( a – d ) Representative WBs from Co-IP experiments investigating the interaction between endogenous STIM1, STIM2 and NR2A, NR2B subunits, demonstrating a change in the interaction upon SOCE activation by TG (TG; +) compared with control neurons treated with 2 mM Ca 2+ (TG; −). Neuronal lysates (Input) and eluted fractions (immunoprecipitates; IP) were separated on 10% sodium dodecyl sulfate gels, analyzed by WB and stained with the corresponding antibody anti-STIM1, STIM2, NR2A, and NR2B (as indicated on the right) as described in “Methods and Materials” section. Anti-IgG antibody was used as a negative control. WB analysis of 40 µg of cell lysate inputs is shown. Molecular weights of the markers run on the same gel are indicated on the left (in kDa). ( b , d ) Unlabeled bands are irrelevant to this experiment. Unspecific IgG band is visible. ( c ) The middle panel shows the WB stained with STIM2 protein after stripping the membrane blotted with anti-STIM1 antibody, indicated with double bands here. ( e ) Pooled data shows a significant change in interaction between STIM proteins and NMDAR subunits after TG treatment. Histogram represents the quantification of STIM-NR (light-green columns) or NR-STIM (dark-green columns) association in neurons incubated in TG compared to neurons incubated in 2 mM Ca 2+ (blue column). Bands of co-immunoprecipitates were analyzed densitometrically and normalized to the level of the loading control (i.e., bands obtained after WB with the antibody used for immunoprecipitation). The results are expressed as a percentage of control (i.e., protein association in 2 mM Ca 2+ ). Bar graphs are mean ± SEM of at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 significantly different compared with control; ns , not significant compared with the control (Mann–Whitney U test).

Article Snippet: The precipitated proteins were then washed three times with repeated centrifugation, eluted in 50 µL of 2× Laemmli Buffer, and subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) and western blot analysis with the indicated primary antibodies rabbit STIM1 (1:200, ProteinTech Group), rabbit STIM2 (1:100, Alomone Labs), rabbit NR2A (1:200, Merck Millipore) and rabbit NR2B (1:300, ProteinTech Group) at 4 °C overnight and then with the appropriate horseradish peroxidase-conjugated secondary antibody IgG (1:5000, Sigma) diluted in blocking solution (TBST: 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20 plus 5% dry non-fat milk).

Techniques: Co-Immunoprecipitation Assay, Activation Assay, Staining, Negative Control, Stripping Membranes, Incubation, Immunoprecipitation, MANN-WHITNEY

Journal: Cell reports

Article Title: Quantitative In Vivo Proteomics of Metformin Response in Liver Reveals AMPK-Dependent and -Independent Signaling Networks

doi: 10.1016/j.celrep.2019.10.117

Figure Lengend Snippet: (A) Clustal alignments of STIM1 S257, S512, S521, and STIM2 S261, S346, S680 across species showing conservation of AMPK consensus motif at identified potential sites of regulation.

Article Snippet: METHOD DETAILS Antibodies and reagents Cell Signaling Antibodies used at 1:1000 in 5% BSA in TBS-T: P-AMPKα Thr172 (#2535), AMPK α1/2 (#2532), P-ACC Ser79 (#3661), ACC (#3662), P-4EBP1 Ser65 (#9452), 4EBP1 (#9452), GAPDH (#5174), LKB1 (#3047), Stim1 (#5668), Stim2 (#4917), AMPK pMOTIF (5759), Myc (#2272), Rabex5 (#7622), P-PKD Ser744/748 (#2054), P-PKD Ser916 (#2051), PKD (#2052), P-JNK Thr183/Tyr185 (#4668), JNK (#9252), GST (#2622), 14-3-3ζ/δ (#7413), P-Raptor Ser792 (#2083), Raptor (#2280).

Techniques:

Journal: Cell reports

Article Title: Quantitative In Vivo Proteomics of Metformin Response in Liver Reveals AMPK-Dependent and -Independent Signaling Networks

doi: 10.1016/j.celrep.2019.10.117

Figure Lengend Snippet: (A) Heatmap of Gene Ontology process: calcium ion binding, purple arrows indicate STIM1 and STIM2.

Article Snippet: METHOD DETAILS Antibodies and reagents Cell Signaling Antibodies used at 1:1000 in 5% BSA in TBS-T: P-AMPKα Thr172 (#2535), AMPK α1/2 (#2532), P-ACC Ser79 (#3661), ACC (#3662), P-4EBP1 Ser65 (#9452), 4EBP1 (#9452), GAPDH (#5174), LKB1 (#3047), Stim1 (#5668), Stim2 (#4917), AMPK pMOTIF (5759), Myc (#2272), Rabex5 (#7622), P-PKD Ser744/748 (#2054), P-PKD Ser916 (#2051), PKD (#2052), P-JNK Thr183/Tyr185 (#4668), JNK (#9252), GST (#2622), 14-3-3ζ/δ (#7413), P-Raptor Ser792 (#2083), Raptor (#2280).

Techniques: Binding Assay

Journal: Cell reports

Article Title: Quantitative In Vivo Proteomics of Metformin Response in Liver Reveals AMPK-Dependent and -Independent Signaling Networks

doi: 10.1016/j.celrep.2019.10.117

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: METHOD DETAILS Antibodies and reagents Cell Signaling Antibodies used at 1:1000 in 5% BSA in TBS-T: P-AMPKα Thr172 (#2535), AMPK α1/2 (#2532), P-ACC Ser79 (#3661), ACC (#3662), P-4EBP1 Ser65 (#9452), 4EBP1 (#9452), GAPDH (#5174), LKB1 (#3047), Stim1 (#5668), Stim2 (#4917), AMPK pMOTIF (5759), Myc (#2272), Rabex5 (#7622), P-PKD Ser744/748 (#2054), P-PKD Ser916 (#2051), PKD (#2052), P-JNK Thr183/Tyr185 (#4668), JNK (#9252), GST (#2622), 14-3-3ζ/δ (#7413), P-Raptor Ser792 (#2083), Raptor (#2280).

Techniques: Recombinant, Sequencing, Modification, Protease Inhibitor, Software, Irradiation