The anti-ezrin mAb did not recognize its epitope on the surface of D54 cells (not shown)

The anti-ezrin mAb did not recognize its epitope on the surface of D54 cells (not shown). specifically identified the eluted gp120 binding proteins confirming their recognition. Ezrin and moesin are structural proteins binding to the cellular membrane and to several cytoskeletal and transmembrane proteins. Our results suggest that ezrin and moesin might play a role as gp160/gp120 binding proteins during the uptake, the assembly or the budding of HIV. after which the supernatant cytosolic and membrane proteins were separated by a high speed centrifugation step (2.5 h, 100?000 em g /em ). The producing membrane pellet was resuspended in sucrose (10%)-comprising BB and the proteins solubilized with 3% w/v octylglucoside. Membrane proteins were approved over an affinity matrix which was prepared by coupling recombinant HIV-1(IIIB)-gp120 (rgp120, AGMED) to CNBr-activated Sepharose 4B beads (Pharmacia). The proteins were passed on the affinity column in TEO buffer (10 mM Tris, pH 8.3, 1 mM EDTA, 0.1% OG) at 4C and allowed to bind for at least 16 h. After washing the column in TEO to remove non-specifically attached proteins, bound proteins were eluted having a sodium chloride gradient. The specificity of the gp120-binding activity of the proteins eluted from your affinity column was confirmed by an immunoblot overlay assay. The GDC-0339 affinity purified proteins were separated on SDS-PAGE, electroblotted onto nitrocellulose (NC) and each lane was incubated with 250 ng rgp120 for 4 h at RT. The immobilized protein-gp120 complexes were detected by a polyclonal anti-gp120 serum and HRP-coupled secondary antibodies. The gp120-overlay was performed in Rabbit Polyclonal to MRC1 TEO-buffer, which was also utilized for binding the proteins to the affinity matrix. Recombinant gp120 bound to proteins of apparent MW of 81 and 76 kDa, but not to the 42 and 38 kDa proteins (Fig. 3 ). In GDC-0339 addition we tested whether gp120 might bind to a p81 and/or p76 connected lipid or glycolipid as for example Gal-S. We performed an immunoblot assay after having subjected these proteins to lipid extraction. No difference between the blots was observed (not shown). Consequently, a lipid-based connection between gp120 and the proteins p81 and p76 can be excluded. These results suggest that gp120 interacts directly with the 81 and 76 kDa proteins. Open in a separate windowpane Fig. 3 Immunoblot-overlay assay of the affinity purified protein portion with rgp120. The affinity purified proteins were separated by SDS-PAGE and transferred to nitrocellulose. Staining of total proteins (lane 2: india ink). Pieces of nitrocellulose were incubated with gp120 binding (250 em /em g/lane) in TEO (10 mM Tris, pH 8.3, 1 mM EDTA, 0.1% OG) at 4C overnight. After incubation with an anti-gp120 serum (1:250 in TBST/BSA) at RT for 4 h, and HRP-conjugated anti-rabbit serum, bands were visualized with chloronaphthol (lane 4: rgp120). As control the rgp120 was omitted (lane 3: control). The two 81 kDa and 76 kDa proteins binding gp120 and the protein with the app. MW of 38 kDa were recognized by amino acid sequence analysis. For this purpose the proteins were digested with trypsin in the gel matrix, the producing fragments extracted and separated by RP-HPLC prior to Edman degradation. The sequence data were confirmed by mass spectrometry (Table 1 ). The 81 kDa protein was unequivocally identified as ezrin following analysis of two peptide sequences. For the 76 kDa protein, a sequence was identified which recognized it as a member of the ERM (ezrin-radixin-moesin; Sato et al., 1992) family of proteins although conclusive recognition was made using methods explained below. In the full case of the 38 kDa protein, four peptide sequences had been identified that have been produced from the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, EC 1.2.1.12). Desk 1 Evaluation of HPLC-purified tryptic peptides GDC-0339 produced from p81, p76 and p38 thead th colspan=”2″ rowspan=”1″ Protein series /th th rowspan=”1″ colspan=”1″ Defined as /th th rowspan=”1″ colspan=”1″ Peptide placement /th th rowspan=”1″ colspan=”1″ Calc. mass /th th rowspan=”1″ colspan=”1″ Exp. mass /th /thead p81SGYLSSEREzrin, individual142C150897.94897.7LIPQREzrin, individual151C155625.77625.6p76LFFLQVKEzrin, individual100C106894.12n.d.Moesin, individual100C106Radixin, individual101C107Merlin, individual117C123p38FHGTVKGAPDH, individual055C060687.80687.4LTGMAFRGAPDH, individual227C233810.98a810.5LEKPAKYDDIKKGAPDH, individual248C2591447.691447.7VVDLMAHMASKEGAPDH, individual323C3341362.59a1362.3 Open up in another window n.d., not really motivated. Using the SWISS-PROT protein series data source p38 was defined as glyceraldehyde-3-phosphate-dehydrogenase (G3P1-Individual or G3P2_Individual), p76 as an associate from the ERM-family (find text message), and p81 as individual ezrin (EZRI_Individual). For N-terminal amino acidity sequencing up to ten Coomassie-blue stained protein rings had been GDC-0339 excised from L?mmli slab gels as well as the protein was digested in the gel matrix with trypsin (1 em /em g for the 81 and 76 kDa proteins and 2 em /em g for the 38 kDa protein) as defined by Eckerskorn and Lottspeich (Eckerskorn and Lottspeich, 1989). The causing peptides had been eluted in the gel and seperated by reverse-phase.