orai1 coding sequences  (Addgene inc)

Journal: Biochemical and biophysical research communications

Article Title: Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein.

doi: 10.1016/j.bbrc.2016.08.072

Figure Lengend Snippet: Fig. 1. Generation of Jurkat cell lines stably producing APP. (A) Immunoblot of endogenous APP in Jurkat, 293T/17, and HeLa cells carrying APP-targeting shRNA (APP KD) or control shRNA. (B) qPCR analysis of relative APP mRNA levels in cells presented in (A). Differences from the reference level of 1 set for HeLa control cells were analyzed using one-sample t-test (n ¼ 4 each). Significant differences are marked with asterisks (***p < 0.001). (C) Immunoblot of APP, Orai1, and STIM1 in Jurkat cells stably expressing APP, APPSWE or an empty cassette (control), compared with wildtype HeLa cells. (D) FACS analysis of Y188-labeled Jurkat APP cells and APPSWE cells. Y188-labeled and mock-labeled 293T/17 cells were analyzed in parallel to set a threshold for Y188- positive cells (horizontal line). The x-axis of both histograms shows the Y188 signal normalized to cell size (FL1-H/FSC) on a logarithmic scale.

Article Snippet: Human APP and ORAI1 coding sequences originated from Addgene plasmids #30137, #30145 and #22754, and were cloned MluI-NotI into a modified pLVTH vector (Addgene #12262), in which eGFP and the H1 promoter were replacedwith an IRES-PURO-WPRE cassette.

Techniques: Stable Transfection, Western Blot, shRNA, Control, Expressing, Labeling

Journal: Biochemical and biophysical research communications

Article Title: Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein.

doi: 10.1016/j.bbrc.2016.08.072

Figure Lengend Snippet: Fig. 2. Microscopic analysis of SOCE in APP-producing Jurkat cells. (A) SOCE measurements in Fura-4F-loaded Jurkat cells that produced APP (n ¼ 13), APPSWE (n ¼ 12) and control cells with an empty cassette (n ¼ 13). (B) SOCE measurements in Jurkat cells that produced dominant-negative Orai1 (Orai1 DN; n ¼ 5) and control cells (n ¼ 5). Shown are averaged traces from representative experiments and calculated mean values with standard error. Differences from control cells were analyzed using unpaired t-test, and significant dif- ferences are marked with asterisks (***p < 0.001).

Article Snippet: Human APP and ORAI1 coding sequences originated from Addgene plasmids #30137, #30145 and #22754, and were cloned MluI-NotI into a modified pLVTH vector (Addgene #12262), in which eGFP and the H1 promoter were replacedwith an IRES-PURO-WPRE cassette.

Techniques: Produced, Control, Dominant Negative Mutation

Journal: Biochemical and biophysical research communications

Article Title: Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein.

doi: 10.1016/j.bbrc.2016.08.072

Figure Lengend Snippet: Fig. 3. Analysis of SOCE in Jurkat cells producing C99 fragments. (A) Immunoblot of small APP fragments (detected with Y188 antibody) in Jurkat cells stably expressing APPSWE, C99 cDNA, or an empty cassette (control) compared with wildtype HeLa cells. DAPT treatment (5 mM for 5 h) is indicated below the gels. (B) Immunoblot of APP, Orai1, and STIM1 in Jurkat cells stably expressing C99 cDNA, C99IND cDNA, or an empty cassette (control) compared with wildtype HeLa cells. (C) qPCR analysis of ORAI1 and STIM1 mRNA levels in C99- producing Jurkat cells relative to those in Jurkat control cells. Differences were analyzed using one-sample t-test (n ¼ 3 each). (D) SOCE measurements in Fura-4F-loaded Jurkat C99 cells (n ¼ 11), C99IND cells (n ¼ 11) and control cells (n ¼ 12). Traces are from representative experiments. The data were analyzed using unpaired t-test.

Article Snippet: Human APP and ORAI1 coding sequences originated from Addgene plasmids #30137, #30145 and #22754, and were cloned MluI-NotI into a modified pLVTH vector (Addgene #12262), in which eGFP and the H1 promoter were replacedwith an IRES-PURO-WPRE cassette.

Techniques: Western Blot, Stable Transfection, Expressing, Control

Journal: Biochemical and biophysical research communications

Article Title: Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein.

doi: 10.1016/j.bbrc.2016.08.072

Figure Lengend Snippet: Fig. 4. Analysis of SOCE in HeLa cells depleted of APP. (A) SOCE measurements in Fura-2-loaded HeLa cells stably expressing APP-targeting shRNA (APP KD; n ¼ 8), ORAI1-targeting shRNA (ORAI1 KD; n ¼ 3) and control shRNA (control; n ¼ 8). The stores were depleted with 20 mM CPA in Ca2þ-free buffer containing 0.5 mM EGTA for 400 s, and SOCE was initiated by the re-addition of 2 mM Ca2þ. Shown are averaged (black) and individual (gray) SOCE traces from representative experiments. The data were analyzed using unpaired t- test, and significant differences from controls are marked with asterisks (***p < 0.001). (B) qPCR analysis of ORAI1 and STIM1 mRNAs in cells presented in (A). Differences from the reference level of 1 set for controls were analyzed using one-sample t-test (n ¼ 3 each). Statistical significance is marked with asterisks (***p < 0.001).

Article Snippet: Human APP and ORAI1 coding sequences originated from Addgene plasmids #30137, #30145 and #22754, and were cloned MluI-NotI into a modified pLVTH vector (Addgene #12262), in which eGFP and the H1 promoter were replacedwith an IRES-PURO-WPRE cassette.

Techniques: Stable Transfection, Expressing, shRNA, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: POST, partner of stromal interaction molecule 1 (STIM1), targets STIM1 to multiple transporters

doi: 10.1073/pnas.1117231108

Figure Lengend Snippet: POST binds Orai1 and is expressed in the ER and plasma membrane. (A) HA epitope-tagged POST specifically binds myc-tagged Orai1 coexpressed in HEK 293 cells. Cell lysates were immunoprecipitated with HA-agarose and stained on Western blot (WB) with myc- or HA-antibody conjugated to HRP. (B) Endogenous POST binds Orai1. Jurkat cell lysates were immunoprecipitated with Orai1, POST antibody, or preimmune rabbit IgG, and probed on WB with the indicated antibody (TrueBlot). Lysates of HEK 293 expressing myc-Orai1 (labeled as myc-Or1) and HA-POST were used as markers. Note that store depletion (cells treated with 1 μM thapsigargin for 10 min in Ca2+-free Ringer's solution) did not affect POST-Orai1 interaction. (C) Live confocal fluorescence images of HEK 293 cells coexpressing POST-GFP and Cherry-STIM1. POST was localized in the cell periphery and intracellular membrane network, where it precisely colocalized with Cherry STIM1 (ER). Note the fraction of POST that is expressed in the cell periphery, where STIM1 is not expressed.

Article Snippet: In-frame subcloning of the human Orai1 coding sequence ( {"type":"entrez-nucleotide","attrs":{"text":"NM_032790","term_id":"170932551","term_text":"NM_032790"}} NM_032790 ; Origene TC124465) into the ATC-TAP vector generated the N-terminal TAP-Orai1 cDNA.

Techniques: Clinical Proteomics, Membrane, Immunoprecipitation, Staining, Western Blot, Expressing, Labeling, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: POST, partner of stromal interaction molecule 1 (STIM1), targets STIM1 to multiple transporters

doi: 10.1073/pnas.1117231108

Figure Lengend Snippet: POST abundance does not substantially affect store-operated Ca2+ influx via Orai1. (A) Store depletion-induced Ca2+ influx in Fura-2–loaded Jurkat cells. siRNA-mediated POST down-regulation (Fig. S6) caused a minor but statistically significant change in Ca2+ influx. (Left) Example and protocol for Fura-2 fluorescence recording. (Right) Average maximal response ± SEM [total of 270 cells for each nonsilencing (NS) and POST siRNA; 5 samples, 2 independent experiments]. ***P < 0.001 (Student's t test). (B) POST overexpression in HEK 293 cells stably transfected with STIM1 did not induce Ca2+ influx and did not modulate store-operated Ca2+ influx via Orai1. Cytosolic Ca2+ changes were recorded using Fura-2. (Inset) Example and protocol of Fura-2 fluorescence recording. The left arrow indicates perfusion with Ca2+-free Ringer's solution plus 1 μM thapsigargin (TG); the right arrow indicates perfusion with Ringer's solution containing 2 mM Ca2+ and 1 μM TG. Bars represent average values of maximal response ± SD to 2 mM Ca2+ (for each condition, 70–80 cells recorded; 3 experiments). The white line indicates average F340/F380 ratio for Ca2+-free Ringer's solution. (C) Representative current-voltage traces (Left) and summary of inward Ca2+ currents measured at −100 mV (Right) from HEK 293T cells overexpressing STIM1, Orai1, and POST. Cells labeled as STIM1-expressing are stable STIM1 transfectants. Currents were measured with 20 mM external [Ca2+] following passive store depletion with 10 mM BAPTA in the pipette. Background currents subtracted for the representative traces and averages (±SEM) are shown in the bar graph.

Article Snippet: In-frame subcloning of the human Orai1 coding sequence ( {"type":"entrez-nucleotide","attrs":{"text":"NM_032790","term_id":"170932551","term_text":"NM_032790"}} NM_032790 ; Origene TC124465) into the ATC-TAP vector generated the N-terminal TAP-Orai1 cDNA.

Techniques: Fluorescence, Over Expression, Stable Transfection, Transfection, Labeling, Expressing, Transferring

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: POST, partner of stromal interaction molecule 1 (STIM1), targets STIM1 to multiple transporters

doi: 10.1073/pnas.1117231108

Figure Lengend Snippet: POST binds Orai1 and is expressed in the ER and plasma membrane. (A) HA epitope-tagged POST specifically binds myc-tagged Orai1 coexpressed in HEK 293 cells. Cell lysates were immunoprecipitated with HA-agarose and stained on Western blot (WB) with myc- or HA-antibody conjugated to HRP. (B) Endogenous POST binds Orai1. Jurkat cell lysates were immunoprecipitated with Orai1, POST antibody, or preimmune rabbit IgG, and probed on WB with the indicated antibody (TrueBlot). Lysates of HEK 293 expressing myc-Orai1 (labeled as myc-Or1) and HA-POST were used as markers. Note that store depletion (cells treated with 1 μM thapsigargin for 10 min in Ca2+-free Ringer's solution) did not affect POST-Orai1 interaction. (C) Live confocal fluorescence images of HEK 293 cells coexpressing POST-GFP and Cherry-STIM1. POST was localized in the cell periphery and intracellular membrane network, where it precisely colocalized with Cherry STIM1 (ER). Note the fraction of POST that is expressed in the cell periphery, where STIM1 is not expressed.

Article Snippet: In-frame subcloning of the human Orai1 coding sequence ( {"type":"entrez-nucleotide","attrs":{"text":"NM_032790","term_id":"170932551","term_text":"NM_032790"}} NM_032790 ; Origene TC124465) into the ATC-TAP vector generated the N-terminal TAP-Orai1 cDNA.

Techniques: Immunoprecipitation, Staining, Western Blot, Expressing, Labeling, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: POST, partner of stromal interaction molecule 1 (STIM1), targets STIM1 to multiple transporters

doi: 10.1073/pnas.1117231108

Figure Lengend Snippet: POST abundance does not substantially affect store-operated Ca2+ influx via Orai1. (A) Store depletion-induced Ca2+ influx in Fura-2–loaded Jurkat cells. siRNA-mediated POST down-regulation (Fig. S6) caused a minor but statistically significant change in Ca2+ influx. (Left) Example and protocol for Fura-2 fluorescence recording. (Right) Average maximal response ± SEM [total of 270 cells for each nonsilencing (NS) and POST siRNA; 5 samples, 2 independent experiments]. ***P < 0.001 (Student's t test). (B) POST overexpression in HEK 293 cells stably transfected with STIM1 did not induce Ca2+ influx and did not modulate store-operated Ca2+ influx via Orai1. Cytosolic Ca2+ changes were recorded using Fura-2. (Inset) Example and protocol of Fura-2 fluorescence recording. The left arrow indicates perfusion with Ca2+-free Ringer's solution plus 1 μM thapsigargin (TG); the right arrow indicates perfusion with Ringer's solution containing 2 mM Ca2+ and 1 μM TG. Bars represent average values of maximal response ± SD to 2 mM Ca2+ (for each condition, 70–80 cells recorded; 3 experiments). The white line indicates average F340/F380 ratio for Ca2+-free Ringer's solution. (C) Representative current-voltage traces (Left) and summary of inward Ca2+ currents measured at −100 mV (Right) from HEK 293T cells overexpressing STIM1, Orai1, and POST. Cells labeled as STIM1-expressing are stable STIM1 transfectants. Currents were measured with 20 mM external [Ca2+] following passive store depletion with 10 mM BAPTA in the pipette. Background currents subtracted for the representative traces and averages (±SEM) are shown in the bar graph.

Article Snippet: In-frame subcloning of the human Orai1 coding sequence ( {"type":"entrez-nucleotide","attrs":{"text":"NM_032790","term_id":"170932551","term_text":"NM_032790"}} NM_032790 ; Origene TC124465) into the ATC-TAP vector generated the N-terminal TAP-Orai1 cDNA.

Techniques: Fluorescence, Over Expression, Stable Transfection, Transfection, Labeling, Expressing, Transferring