Journal: The Journal of Cell Biology

Article Title: STIM1, an essential and conserved component of store-operated Ca 2 + channel function

doi: 10.1083/jcb.200502019

Figure Lengend Snippet: The effect of STIM1 suppression on Ca 2 + signaling in individual Jurkat T cells. (A) Western blot of 4A5 cell lysates (lane 2), compared with control 2A4 cells (lane 1) showing >50% reduction in STIM1 protein, with no change in the protein levels of GAPDH. (B) The specificity of STIM1 suppression was confirmed by RT-PCR analysis showing a reduction in STIM1, but not STIM2 or GAPDH, mRNA levels in 4A5 cells (lane 2) compared with control 2A4 cells (lane 1). (C) Intracellular Ca 2+ responses in 51 Jurkat 2A4 control cells. Cells were bathed in Jurkat Ringer (2 mM Ca 2+ ), low-Ca 2+ (0.4 mM) Jurkat Ringer, and Ca 2+ -free Jurkat Ringer with 1 μM TG, as indicated. The first peak is due to Ca 2+ release from internal stores in the presence of TG. The second and third peaks result from Ca 2+ entry through CRAC channels upon addition of 0.4 and 2 mM external Ca 2+ , respectively. Sustained [Ca 2+ ] i was measured 5 min after readdition of 2 mM external Ca 2+ . (D) Averaged [Ca 2+ ] i in control 2A4 cells from the same experiment. (E) Intracellular Ca 2+ responses in 40 STIM1-suppressed 4A5 Jurkat cells. (F) Averaged [Ca 2+ ] i in STIM1-suppressed 4A5 cells from the same experiment as in D. (G) Combined data from three control experiments (164 cells, white bars) and three experiments with STIM1-suppressed cells (141 cells, gray bars). Averaged values of peak and sustained [Ca 2+ ] i are significantly different in STIM1-suppressed cells (P < 8 × 10 −6 , < 8 × 10 −6 , and < 2 × 10 −5 , respectively, by independent two populations t test). (H) Maximal rate of Ca 2+ rise upon Ca 2+ readdition as an estimate of Ca 2+ influx. Representative averaged traces obtained in the same experiments as in A–D are shown (control 2A4 cells, closed squares; STIM1-suppressed 4A5 cells, open squares), along with corresponding differentiated [Ca 2+ ] i traces, d[Ca 2+ ] i /dt (right axis), for control 2A4 cells (black line without symbols) and STIM1-suppressed 4A5 cells (gray line). The peak derivatives correspond to the maximal rate of Ca 2+ rise in nM. (I) STIM1 expression and d[Ca 2+ ] i /dt. The maximal rate of [Ca 2+ ] i rise after 0.4 mM or 2 mM Ca 2+ readdition in control 2A4 cells (white bars: 164 cells in three experiments); and STIM1-suppressed 4A5 cells (gray bars: 141 cells in three experiments; P < 3 × 10 −7 or < 2 × 10 −7 for 0.4 and 2 mM Ca 2+ , respectively).

Article Snippet: STIM1 pAbs (1:2,500) were generated by Alpha Diagnostic, Inc. to a COOH-terminal peptide (STIM1-CT: DNGSIGEETDSSPGRKKFPLKIFKKPLKK) as described previously ( ) and were used for Western blots.

Techniques: Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: The Journal of Cell Biology

Article Title: STIM1, an essential and conserved component of store-operated Ca 2 + channel function

doi: 10.1083/jcb.200502019

Figure Lengend Snippet: Reduction of Jurkat CRAC current by STIM1 suppression. (A) Current development in selected control 2A4 Jurkat cell. Cells were first bathed in Ca 2+ -free Jurkat external solution for 5 min and dialyzed with BAPTA-containing Jurkat internal solution. CRAC current was revealed after exchange of Ca 2+ -free Jurkat solution to 20 mM Ca 2+ Jurkat external solution. Maximal current density was evaluated at −110 mV. (B) Leak-subtracted current-voltage relationship of fully developed CRAC current recorded in the same control 2A4 Jurkat cell. (C) Suppression of CRAC current in STIM1-suppressed 4A5 Jurkat cell. (D) Leak-subtracted I-V relationship in the same cell, as in C. (E) CRAC current density in control 2A4 cells (circles, n = 11) and STIM1-suppressed 4A5 cells (squares, n = 11). Horizontal lines indicate the mean value of current density in each group. (P < 3 × 10 −6 ).

Article Snippet: STIM1 pAbs (1:2,500) were generated by Alpha Diagnostic, Inc. to a COOH-terminal peptide (STIM1-CT: DNGSIGEETDSSPGRKKFPLKIFKKPLKK) as described previously ( ) and were used for Western blots.

Techniques: Control

Journal: The Journal of Cell Biology

Article Title: STIM1, an essential and conserved component of store-operated Ca 2 + channel function

doi: 10.1083/jcb.200502019

Figure Lengend Snippet: Suppression of STIM1 in HEK293 cells inhibits SOC influx. (A) RT-PCR analysis. STIM1 and STIM2 mRNA levels were reduced in cells transfected with the appropriate siRNA to <50% of control cells (transfected with scrambled siRNA). GAPDH levels were unchanged in either treatment group. (B) Western blot analysis. In cells transfected with the STIM1 siRNA, STIM1 protein levels were reduced to <10% of control levels, whereas GAPDH levels were unchanged. (C) Immunofluorescence localization of STIM1 in HEK293 cells. Nuclear staining pattern (left) with DAPI (Molecular Probes) in HEK293 cells treated with either a scrambled siRNA (top) or siRNA to STIM1 (bottom). No change in nuclear staining pattern or intensity was observed after RNAi-induced suppression of STIM1. STIM1-associated immunofluorescence (right) in HEK293 cells treated with either control (top) or STIM1 (bottom) siRNAs. In control cells, STIM1 has a diffuse reticulated localization pattern with some punctuate staining, which is consistent with expression associated with plasma membrane and ER. The intensity of STIM1 immunofluorescence was markedly decreased in the cells treated with STIM1 siRNA. (D) Calcium signals in HEK293 cells after RNAi-mediated knockdown. Suppression of STIM1 (dotted line) reduced SOC influx by 60% compared with control (solid line; P < 10 −4 , unpaired t test), whereas suppression of STIM2 (dashed line) had little effect. Data indicate RFUs in 384-well plates monitored in a FLIPR 384 fluorimeter. The traces are from a representative experiment, and are averaged signals from 48 wells per group. Traces from cells treated with vehicle (DMSO) instead of TG were essentially flat (not depicted for clarity). (E) Calcium signals after muscarinic receptor activation. RT-PCR analysis revealed that the muscarinic receptor, subtype m3, is expressed in our HEK293 cells (not depicted). 300 μM of methylcholine evoked Ca 2+ -release transients in Ca 2+ -free buffer were not inhibited by STIM1 suppression, but SOC influx upon readdition of 2 mM Ca 2+ was greatly reduced in STIM1 siRNA-treated cells (dotted line) compared with control cells (solid line). The apparent enhancement of the methylcholine-evoked Ca 2+ release transient in the STIM1-suppressed cells was not a consistent finding. (F) TG-induced Ba 2+ entry. The rate of TG-induced Ba 2+ entry in STIM1-suppressed cells (dotted line) was significantly lower than in control cells (solid line) or STIM2-suppressed cells (dashed line; P < 10 −4 , unpaired t test).

Article Snippet: STIM1 pAbs (1:2,500) were generated by Alpha Diagnostic, Inc. to a COOH-terminal peptide (STIM1-CT: DNGSIGEETDSSPGRKKFPLKIFKKPLKK) as described previously ( ) and were used for Western blots.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Western Blot, Immunofluorescence, Staining, Expressing, Clinical Proteomics, Membrane, Knockdown, Activation Assay

Journal: The Journal of Cell Biology

Article Title: STIM1, an essential and conserved component of store-operated Ca 2 + channel function

doi: 10.1083/jcb.200502019

Figure Lengend Snippet: Overexpression of STIM1 in HEK293 cells. (A) Western blot analysis of STIM1 (top band) and GAPDH (bottom band) proteins in HEK-STIM1 (STIM1 overexpressing) cells and control cells (HEK-Zeo cells); lane 1, 10 μg protein; lane 2, 1 μg protein. We estimate STIM1 protein levels to be nearly 100-fold greater than in the HEK-STIM1 cells compared with control cells. (B) Representative traces of TG-induced Ca 2+ release and TG-induced Ca 2+ entry in HEK-STIM1 cells (dotted line) compared with HEK-Zeo cells (solid line). TG-induced Ca 2+ entry was enhanced in HEK-STIM1 cells by an average of 17% in four experiments. (C and E) Time course of outward current at +80 mV and inward current at −110 mV (note different scales) for control HEK-Zeo (E) and HEK-STIM1 cells (C). (D and F) Representative current-voltage relationships immediately after break-in to achieve whole-cell recording and 5 min later in control HEK-Zeo (D) and HEK-STIM1 cells (F). Outwardly rectifying Mg 2+ -inhibited cation current representing channel activity of TRPM7 disappeared as Mg 2+ diffused into the cell from the pipette. (G and H) Inward and outward currents were not significantly altered by overexpression of STIM1; n = 10 cells for each group. Error bars represent SEM.

Article Snippet: STIM1 pAbs (1:2,500) were generated by Alpha Diagnostic, Inc. to a COOH-terminal peptide (STIM1-CT: DNGSIGEETDSSPGRKKFPLKIFKKPLKK) as described previously ( ) and were used for Western blots.

Techniques: Over Expression, Western Blot, Control, Activity Assay, Transferring

Journal: The Journal of Cell Biology

Article Title: STIM1, an essential and conserved component of store-operated Ca 2 + channel function

doi: 10.1083/jcb.200502019

Figure Lengend Snippet: Specificity of STIM1 RNAi in SH-SY5Y cells. (A) Effects of STIM1 RNAi on SOC influx in SH-SY5Y cells. TG-dependent Ca 2+ entry in STIM1 siRNA-treated cells (dotted line) is greatly reduced compared with control cells (solid line). Traces from cells treated with vehicle (DMSO) instead of TG were essentially flat (not depicted for clarity). (B) KCl-evoked Ca 2+ signals as a measure of voltage-gated Ca 2+ channel activity. Data presented as fluo-4 RFUs. At concentrations of 3 mM KCl or below, no significant change in cytosolic Ca 2+ was observed. At 10, 20, and 60 mM KCl, a rapid rise in cytosolic Ca 2+ was detected. (C) Maximal KCl-evoked RFU values are not different in control and STIM1-knockdown cells. (D) STIM1 suppression does not affect the resting membrane potential or the response to depolarization in SH-SY5Y cells. To monitor changes in membrane potential, a FLIPR membrane potential assay kit (Molecular Devices) was used as per the manufacturer's protocols. Data presented in RFUs. Cells were depolarized with increasing concentration of KCl, as in B. Error bars represent SD.

Article Snippet: STIM1 pAbs (1:2,500) were generated by Alpha Diagnostic, Inc. to a COOH-terminal peptide (STIM1-CT: DNGSIGEETDSSPGRKKFPLKIFKKPLKK) as described previously ( ) and were used for Western blots.

Techniques: Control, Activity Assay, Knockdown, Membrane, Concentration Assay