IGFBP3 Antibody - #AF6203

Figure 2 Orai1–3 protein and IGFBP3 expression levels and SOCE activity are significantly increased in HCAECs cultured in HG for 7 days and coronary artery endothelial cells of streptozotocin-induced diabetic mice with no change in STIM1 expression levels; increased Orais promotes proliferation of HCAECs and decreased IGFBP3 reduces proliferation of HCAECs. (A, C) Representative traces and (B, D) summary data showing the maximum increase in SOCE in HG-cultured HCAECs. After treatment of HCAECs with either 2 µM TG or 100 µM ATP for 10 min, application of 2 mM Ca2+ induces significant increase in [Ca2+]i through SOCE. (E, G, I, K, A') Representative western blot images and (F, H, J, L, B'–E') summary data showing Orai1, Orai2, Orai3, STIM1 and IGFBP3 expression levels in HCAECs cultured in NG or HG medium. (M, O, Q, F') Representative images of coronary artery endothelium immunostaining (brown; eg, red arrowheads) for protein expression levels of Orai1–3 and IGFBP3 in a mouse model of DM and control mice. Magnification, ×400. Scale bar, 50 µm, applies to all panels. (N, P, R, G') Quantification of Orai1–3 and IGFBP3 protein expression levels in the coronary artery endothelium of a mouse model of DM and control mice. (S) Representative western blot images and (T) summary data showing PCNA protein expression levels in HCAECs cultured in HG or NG medium for 7 days in the presence or absence of the SOCE agonist ATP (100 µM) or the SOCE inhibitor BTP2 (10 µM). (H') Representative western blot images and (I') summary data showing IGFBP3 siRNA knockdown of PCNA protein level. (U) Viability of HCAECs cultured in NG or HG medium for 7 days in the presence or absence of ATP (100 µM) or BTP2 (10 µM) as detected using the CCK-8 assay. (J') Results of CCK-8 assay examining HCAEC viability after cells were cultured in either NG or HG medium for 7 days and then transfected with either IGFBP3-specific siRNA or scrambled siRNA. β-tubulin or GAPDH was used as loading controls. Values represent mean±SEM (n=4–21 samples). *P<0.05, **P<0.01, ***P<0.001 compared with NG-cultured cells or control groups. BTP2, N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide; CCK-8, Cell Count Kit-8; DM, diabetes mellitus; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCAECs, human coronary artery endothelial cells; HG, high glucose; IGFBP3, insulin-like growth factor binding protein 3; IOD, integrated optical density; NG, normal glucose; PCNA, proliferating cell nuclear antigen; siRNA, small interfering RNA; SOCE, store-operated Ca2+ entry; STIM1, stromal interaction molecule 1; TG, thapsigargin.

Figure 3 Physical interactions between the Orais and IGFBP3 proteins as examined by coimmunoprecipitation and immunofluorescence assays; SOCE regulates the expression level of IGFBP3 protein, which regulates the expression level of the Orai proteins in HCAECs cultured in HG medium. (A, C, E) HCAECs were cultured in HG medium for 7 days and then subjected to immunoprecipitation. Assays positive for Orai1, Orai2, or Orai3 proteins were used to pull down IGFBP3. (G, I, K) For reverse coimmunoprecipitation, immunoprecipitation assays positive for IGFBP3 were used to pull down Orai1, Orai2, or Orai3. (B, D, F, H, J, L) Summary data showing the statistical results of the corresponding coimmunoprecipitations. (M, O, Q) Representative confocal microscopy images showing co-localization analysis of anti-Orai1, 2, or 3 antibodies (red) and anti-IGFBP3 antibody (green). HCAECs were cultured in HG medium for 7 days and then fixed and incubated with the anti-Orai antibodies (red) and the anti-IGFBP3 antibody (green) and imaged using confocal microscopy. Representative confocal microscopy images of each antibody and the final merged images are shown. Nuclei are labeled blue with DAPI. (N, P, R) Fluorescence intensity profiles and summary data of anti-Orai antibodies (red) and the anti-IGFBP3 antibody (green) in the regions delineated by the corresponding yellow line. Scale bar, 10 mm. Co-localization area per cell was quantified using ImageJ. Representative western blot images (A') and summary data (B'–D') showing expression levels of the Orai proteins and SOCE activity induced by 100 µM ATP or 2 µM TG in HCAECs cultured in HG medium for 7 days after transfection with IGFBP3-specific siRNA or scrambled siRNA (siScrambled). Representative traces (E', G') and summary data (F', H') showing ATP-induced or TG-induced SOCE activity in HG-cultured HCAECs. Representative western blot images (I') and summary data (J') of IGFBP3 expression level in HCAECs cultured in HG medium for 7 days after the addition of the SOCE agonist ATP (100 µM) or the SOCE inhibitor BTP2 (10 µM). The same amount of solvent (ATP solvent was ddH2O (deionized distilled water); BTP2 solvent was DMSO (Dimethyl sulfoxide)) was used as control. (K') CCK-8 assay was used to detect the viability of HCAECs transfected with IGFBP3 siRNA (siIGFBP3) and cultured in medium with NG or HG for 7 days and treated with the SOCE agonist ATP (100 µM) or the SOCE inhibitor BTP2 (10 µM). Representative western blot images (L') and summary data (M') showing the expression levels of PCNA protein in HCAECs transfected with IGFBP3 siRNA and cultured in NG or HG medium for 7 days and then treated with ATP (100 µM) or BTP2 (10 µM). Values are mean±SEM (n=3–9 samples). *P<0.05, **P<0.01, ***P<0.001 compared with NG-cultured cells or control groups. BTP2, N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide; CCK-8, Cell Count Kit-8; DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCAECs, human coronary artery endothelial cells; HG, high glucose; IGFBP3, insulin-like growth factor binding protein 3; NG, normal glucose; PCNA, proliferating cell nuclear antigen; siRNA, small interfering RNA; SOCE, store-operated Ca2+ entry; TG, thapsigargin.