To detect cytochrome c and cleaved caspase 3, cellular lysis was performed in 30 min on an ice lysis buffer consisting of the following agents: 20 mM of HEPES (pH 7.6), glycerol (20%), 500 mM of NaCl, 1.5 mM of Mg[Cl.sub.2], 0.2 mM of EDTA, Triton X100 (0.1%), 10 [micro]g/mL of aprotinin, 2.5 [micro]g/mL of leupeptin
, 0.5 mM of phenylmethanesulfonyl fluoride, 1 mM of dithiothreitol, and sodium dodecyl sulfate (SDS).
Samples of IGM were homogenized in an ice cold lysis buffer (10 mm Tris-Cl, pH 8.0; 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 40 uM phenylmethylsulfonyl fluoride, and 1 uM leupeptin
Cells were lysed on ice with a lysis solution (50 mM TRIS base, 5 mM EGTA, and 150 mM NaCl, (1% Trion)) containing a cocktail of protease inhibitors (2 mM NaVO4, 50 mM NF, 1 mM PMSF, 20 [micro]M phenylalanine, 10 Mm sodium molybdate, 10 [micro]g/ml leupeptin
, and 8 [micro]g/ml aprotinin), the latter two reagents both being added on day of use.
Cells were harvested and rinsed twice with cold PBS, and 100 [micro]L of RIPA lysis buffer (150 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% Na[N.sub.3], 1 mM EDTA, 1 mM PMSF, 1 [micro]g/mL aprotinin, 1 [micro]g/ mL leupeptin
, 1 [micro]g/mL pepstatin, 1 mM sodium orthovanadate, and 1 mM NaF) was added to each well.
After the transfection process, the cells were lysed in the lysing buffer (50 mM Tris base-hydrochloric acid (Tris-HCl), pH 7.4; 150 mM sodium chloride (NaCl); 1% tergitol-type NP-40; and 0.25% Na-deoxycholate) containing protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF); 1 mM ethylenediaminetetraacetic acid (EDTA); aprotinin, pepstatin, and leupeptin
(1 [micro]g/mL each); 1 mM sodium orthovanadate ([Na.sub.3]V[O.sub.4]); and 1 mM sodium fluoride (NaF)) at 4[degrees]C.
Both buffers were supplemented with 1 mM PMSF (added just prior to addition to cells), 2 mM MgSO4 andbenzonase (500 IU/ml) to digest DNA polymers, 1: 100 diluted Sigma phosphatase inhibitor cocktail (2 and 3) and 1: 1000 diluted Sigma protease inhibitor cocktail, and 10 [micro]g/ml leupeptin
to inhibit proteases and phosphatases that might be released on cell lysis.
, 2',7'-dichlorofluorescin diacetate, lipopolysaccharides (LPS), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT), Nonidet P-40, phenylmethylsulfonyl fluoride (PMSF), RA, sodium fluoride, sodium chloride, sodium phosphate, Tris-HCl, Triton X-100, and Tween-20 were purchased from Sigma-Aldrich (USA).
To prepare tissue lysate, kidney tissue (~50 mg) were homogenized in Tris-HCl buffer (pH - 8.0) containing leupeptin
(10 [micro]g/ml), aprotinin (10 [micro]g/ml), Triton X-100 (1%), PMSF (1 mM), EGTA (1 mM), NaF (5 mM), and sodium orthovanadate (10 mM).
Forty-eight hours after transfection, the cells were scraped from the plates 2 times with 5 ml buffer A (3 mmol [l.sup.-1] MgC12, 140 mmol [l.sup.-1] NaCl, 50 mmol [l.sup.-1] HEPES pH 6.6, aprotinin [10 mg m[l.sup.-1]], leupeptin
[10 mg m[l.sup.-1]]).
5 mmol/L MgCl[sub]2; 1 mmol/L DTT; 5% glycerin; 0.2 mmol/L EDTA; 1 mmol/L PMSF; 3 mg/L aprotinin; 3 mg/L leupeptin
; 2 mg/L pepstainA; 1% NP-40) for 10 min, and then centrifuged on ice (14,000 r/min) for 1 min; the deposit was prepared in 15 [micro]l buffer B on ice (20 mmol/L HEPES, pH 7.9; 420 mmol/L NaCl; 1.5 mmol/L MgCl[sub]2; 0.2 mmol/L EDTA; 0.5 mmol/L DTT; 25% glycerin; 0.5 mmol/L PMSF; 5 mg/L aprotinin; 5 mg/L leupeptin
; 3 mg/L pepstainA) for 30 min, and then centrifuged on ice for 15 min (14,000 r/min).
5% sodium deoxycholate, 10 mg/ml aprotinin, 10 mg/ml leupeptin
, and 1 mM phenylmethylsulfonylfluoride.
Cell lysates were prepared in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM [Na.sub.3]V[O.sub.4], 1 [micro]g/ml leupeptin
, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5).