0 with 0.1 M acetic acid (46); peptide-N4-[N-acetyl-��-glucosaminyl] asparagine amidase 10 U/ml from Flavobacterium selleck kinase inhibitor meningosepticum (N-glycanase; ProZyme; Hayward, CA) for which the sample is first denatured by boiling in 0.2 M NaPO4, 0.5% SDS, and 0.05 M ��-mercaptoethanol, pH 7.5. Nonidet P-40 was added to a sevenfold excess over SDS and N-glycanase treatment proceeds at 37��C for 18 h (48); and pronase (Sigma-Aldrich), from Streptomyces griseus, 1% by weight at 37��C for 72 h, with further additions of enzyme (0.5% by weight) at 24 and 48 h, in 0.1 M Tris?HCl buffer, pH 8.0, containing 1 mM CaCl2 (21). Pronase was preincubated at 60��C to inactivate contaminating enzymes. Following all digestions, chromatography was performed as described above.

Fractions (5 ml) were collected, and an aliquot of each fraction was counted for radioactivity. CsCl density gradient centrifugation. The lyophilized cell culture Vo fractions obtained from different HTGM cultures were treated with hyaluronidase as described above, dialyzed, and lyophilized, and the fractions were subjected to density gradient centrifugation in CsCl adjusted to a density of 1.52 g/ml (51). Centrifugation (32,000 g, 20��C, 72 h) was performed by using a Beckman SW 41 Ti rotor (Beckman Coulter, Brea, CA). Sequential fractions were collected from the bottom of the tube, weighed, and counted for radioactivity. Amino acid analysis. The amino acid compositions of hyaluronidase-treated Vo fractions prepared from HTGM cell cultures from a third specimen and from an organ culture of tracheobronchial submucosal tissue fragments were determined by using a Beckman 6300 amino acid analyzer (Beckman Instruments, Palo Alto, CA) by the method of Bidlingmeyer et al.

(6). Total RNA isolation and RT-PCR. Ten-day-old HTGM cell cultures from three separate individuals were used for the analysis of mucin gene transcripts by RT-PCR for expression of MUC1, 2, 3, 4, 5B, 5AC, 6, 7, 13, 15, 16, 17, 19, and 20 with ��-actin as a quality control (Table 1). Total RNA was isolated and purified according to standard procedure using TRIzol reagent (Invitrogen, Carlsbad, CA). The quality of isolated RNA was determined by agarose gel electrophoresis followed by spectrophotometry. RNA samples were then incubated with oligo(dT) and random primers in a 12-��l reaction volume. The samples were heated to 70��C for 10 min and immediately placed on ice.

A mixture containing 1�� reaction buffer (GIBCO-BRL) and deoxynucleoside triphosphates was added to each sample. Superscript II TM reverse transcriptase was added to the reaction mixture to a final volume of 20 ��l. The reaction was then incubated for 1 h at 42��C and terminated by heating at 70��C for 10 min. Next, the RT samples were used for the mucin gene PCR reactions. The PCR components included 2 ��l of template DNA, 1�� Taq polymerase reaction buffer, 2 ��M of each primer, Carfilzomib 200 ��M of dNTPs, 1% DMSO, and Taq polymerase.