Fig. 5.
A: characteristic Western blots of human airway smooth muscle cells (hASMC) 24 h after exposure to air (lanes 1, 2) or Br2 (100 ppm for 10 min; lanes 3, 4, 5). Cells were immunostained with a specific antibody against the calcium-sensing receptor (Ca-SR; 4 µg/mL), as described in materials and methods. The two bands at 120 and 250 kDa correspond to the monomer and dimer forms of the receptor. This experiment was repeated twice with identical results. B and C: quantification of the 120 kDa and 250 kDa bands in hASMC exposed to air or 24 h postexposure to Br2. Each point represents the digitized value of a single lane; individual values are means ± 1 SE; n = 5 for air and n = 6 for 24 h postexposure to Br2. Statistical analysis was performed with the Student’s t test. D: hASMC were transfected with a scrambled siRNA (scr-siRNA; lanes 1, 2 and 5, 6) or a Ca-SR siRNA (lanes 3, 4, and 7, 8) and exposed to air (lanes 1–4) or Br2 (100 ppm for 10 min; lanes 5–8) and returned to an incubator at 37°C vented with 95% air and 5% CO2 for 24 h. At that time, cells were processed for Western blotting analysis with a Ca-SR antibody as described in materials and methods. There was a significant increase of Ca-SR protein in Br2-exposed cells transfected with scrambled siRNA (lanes 5, 6), but not in those transfected with Ca-SR siRNA (lanes 7, 8). Each blot was repeated twice. An uncropped version of this blot is shown in Fig. 10. E and F: quantification of the 120 and 250 kDa bands separately 24 h after exposure to air or Br2. Values of individual digitized lanes and means ± SE; n = 5 for each condition. G: Western blots of primary mouse airway cells (mpASMC) exposed to air or Br2. Twenty four hours postexposure, cells were processed for Western blotting with a primary antibody against the Ca-SR protein. Each blot was repeated twice. An uncropped version of this blot is shown in Fig. 10. H: quantification of the 120 kDa bands. Individual values and means ± SE; n = 5 for each condition. I: hASMC were transfected with scrambled (scr) or Ca-SR siRNA as described in the text. Vm was measured 1 h after Br2, following the addition of nifedipine (10 µM) or vehicle (DMSO) at 30 min postexposure. Individual values and means ± 1 SE; n = 15 for each condition. Statistical analysis was performed with one-way ANOVA followed by the Tukey t test for multiple comparisons. J: hASMC were transfected with Ca-SR siRNA or a scrambled siRNA 48 h before they were exposed to Br2 (100 ppm for 10 min) or air. Thirty minutes postexposure, nifedipine (10 µM) or vehicle (DMSO) was added and [Ca2+]i was measured using Fura2 as described in the text. Individual values and means ± 1 SE; n = 10 for each condition. Statistical analysis was performed with one-way ANOVA followed by the Tukey t test for multiple comparisons.