Figure 4

A, Upper panel: Immunoblots of the DNP-derivatives of lung proteins of guinea pigs exposed to smoke from 1, 2 and 3 cigarettes. Twenty five μg proteins isolated from air-exposed or CS-exposed guinea pigs fed vitamin C-free diet for 7 days were converted to the DNP-derivative followed by immunoblotting as mentioned in the Methods. Lower panel: The membrane was reprobed with anti PARP antibodies which show that there was no PARP cleavage. B, Detection of DNA strand breaks (TUNEL assay) in lung cells of guinea pigs exposed to smoke from 1, 2 and 3 cigarettes. C, Quantitative evaluation of TUNEL assay and protein carbonyl formation in the lungs of guinea pigs exposed to smoke from 1, 2 and 3 cigarettes. Protein carbonyl was measured by densitometric scanning using Quantity One- 4.4 (Bio-Rad) Software. Bars over the respective columns represent means ± SD (n = 4). D, Western blots of mitochondrial and cytosolic cytochrome c of lungs of guinea pigs exposed to 1, 2 and 3 cigarettes; M represents mitochondria and C, cytosol. E, Immunoblots of VEGF in BALF isolated from guinea pigs with and without exposure to smoke from 3 cigaretes. Lane 1, sham control after exposure to air; lane 2, after exposure to 3 cigarettes; Similar results were obtained from 3 different animals. The upper panel represents the dimmer of VEGF (Mr 42 kDa) and lower panel, the monomer (Mr 21 kDa). Loading control (SDS-PAGE) is shown at the bottom. F, Immunoblots of VEGRF2 in lung tissue of guinea pigs with and without exposure to smoke from 3 cigarettes. Lane 1, sham control after exposure to air; lane 2, after exposure to 3 cigarettes; lower panel indicates loading control. Similar results were obtained from 3 different animals. G, western blots showing levels of MMP-9 and MMP-12 in lung tissue and BALF isolated from guinea pigs fed 1 mg vitamin C/day and exposed to air or CS for 14 days; In each of the blots under MMP-9 and MMP-12, the left blot indicates exposed to air and the right, exposed to CS.