Formed with a Zeiss Photoscope Imager Z I.room temperature. After additional washes with TBST, the membranes were incubated with a secondary antibody conjugated with horseradish peroxidase at 1:5,000 dilution for 1 h and transferred to Vectastain ABC (Vector Laboratories, Burlingame, CA, USA). The protein spots on the film were matched with the 2DE map of the same sample and excised from the 2-DE gel stained with Coomassie Brilliant Blue. The excised proteins were then digested as described previously for protein identification by mass spectrometry using spots from different gel with at 25033180 least two replicates. The obtained peptide mass fingerprint was used to search through the Swiss-Prot and National Center for Biotechnology Information nonredundant databases by the Mascot search engine (www. matrixscience.co.uk). Protein identification was reconfirmed by an ESI-MS/MS approach. The database search was finished with the Mascot search engine (www.matrixscience. co.uk) using a Mascot MS/MS ion search.Cell Viability Analysis by 3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium Bromide (MTT) AssayCell growth inhibition was determined by MTT assay (Sigma). Briefly, myeloma cells (26104 cells/well to 36104 cells/well) were seeded on a 96-well plate at a volume of 100 mL per well and incubated for 24 h. The cells were then treated with normal saline (NS), control rabbit IgG, or PAb for 48 h at 37uC and subjected to MTT assay. Cells treated with NS served as the indicator of 100 cell viability.Two-dimensional Electrophoresis (2D )About 26107 ARH-77 cells were solubilized in 1 mL of lysis solution (7 M urea, 2 M thiourea, 4 CHAPS, 2 mmol/L TBP, 0.2 ampholyte, traces of bromophenol blue) at 4uC for 20 min. Insoluble material was removed by centrifugation at 15000 rpm at 4uC for 30 min. Protein concentrations were determined by the Bradford method. Samples were frozen at 270uC and thawed immediately before use. Approximately 1 mg protein was loaded on 17 cm of IPG Ready Strips. After rehydrating the strips for 14 h, IEF was carried out for 1 h at 200 V, 1 h at 500 V, and 1 h at 1000 V. A gradient was then applied from 1,000 to 8,000 for 1 h and finally at 8,000 V for 8 h to reach a total of 72 KVh at 20uC. After IEF separation, the gel strips were incubated first in equilibration buffer (50 mM MedChemExpress 76932-56-4 Tris-HCl, pH 8.8, 6 M urea, 30 glycerol, 2 SDS) with 10 mg/mL DTT and then in equilibration buffer with 25 mg/mL iodoacetamide for 15 min each. The strips were then loaded on 12 SDS-PAGE gel and electrophoresed for 20 min at a constant current of 10 mA and at 30 mA per gel until the bromophenol blue indicator reached the bottom of the gels. One gel was then stained with Coomassie Brilliant Blue R-250 and destained with 40 HIV-RT inhibitor 1 custom synthesis methanol and 10 acetic acid. Another gel was analyzed by 2D Western blot.Apoptosis Analysis by Flow Cytometry AssayThe cells (36105 per well) were plated in 6-well plates and treated with NS, control IgG (200 mg/mL) or PAb (200 mg/mL). After 48 h, flow cytometric analysis was conducted to identify subG1 phase cells/apoptotic cells. Briefly, the cells were suspended in 1 mL of hypotonic fluorochrome solution containing 50 mg/mL propidium iodide in 0.1 sodium citrate with 0.1 Triton X-100 and analyzed by a flow cytometer. Apoptotic cells appeared in the cell cycle distribution as cells with DNA content less than that of G1 phase cells.Antitumor Effect of PAb on Xenograft SCID Mouse Models with ARH-Human ARH-77 MM cells (56106) were.Formed with a Zeiss Photoscope Imager Z I.room temperature. After additional washes with TBST, the membranes were incubated with a secondary antibody conjugated with horseradish peroxidase at 1:5,000 dilution for 1 h and transferred to Vectastain ABC (Vector Laboratories, Burlingame, CA, USA). The protein spots on the film were matched with the 2DE map of the same sample and excised from the 2-DE gel stained with Coomassie Brilliant Blue. The excised proteins were then digested as described previously for protein identification by mass spectrometry using spots from different gel with at 25033180 least two replicates. The obtained peptide mass fingerprint was used to search through the Swiss-Prot and National Center for Biotechnology Information nonredundant databases by the Mascot search engine (www. matrixscience.co.uk). Protein identification was reconfirmed by an ESI-MS/MS approach. The database search was finished with the Mascot search engine (www.matrixscience. co.uk) using a Mascot MS/MS ion search.Cell Viability Analysis by 3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium Bromide (MTT) AssayCell growth inhibition was determined by MTT assay (Sigma). Briefly, myeloma cells (26104 cells/well to 36104 cells/well) were seeded on a 96-well plate at a volume of 100 mL per well and incubated for 24 h. The cells were then treated with normal saline (NS), control rabbit IgG, or PAb for 48 h at 37uC and subjected to MTT assay. Cells treated with NS served as the indicator of 100 cell viability.Two-dimensional Electrophoresis (2D )About 26107 ARH-77 cells were solubilized in 1 mL of lysis solution (7 M urea, 2 M thiourea, 4 CHAPS, 2 mmol/L TBP, 0.2 ampholyte, traces of bromophenol blue) at 4uC for 20 min. Insoluble material was removed by centrifugation at 15000 rpm at 4uC for 30 min. Protein concentrations were determined by the Bradford method. Samples were frozen at 270uC and thawed immediately before use. Approximately 1 mg protein was loaded on 17 cm of IPG Ready Strips. After rehydrating the strips for 14 h, IEF was carried out for 1 h at 200 V, 1 h at 500 V, and 1 h at 1000 V. A gradient was then applied from 1,000 to 8,000 for 1 h and finally at 8,000 V for 8 h to reach a total of 72 KVh at 20uC. After IEF separation, the gel strips were incubated first in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30 glycerol, 2 SDS) with 10 mg/mL DTT and then in equilibration buffer with 25 mg/mL iodoacetamide for 15 min each. The strips were then loaded on 12 SDS-PAGE gel and electrophoresed for 20 min at a constant current of 10 mA and at 30 mA per gel until the bromophenol blue indicator reached the bottom of the gels. One gel was then stained with Coomassie Brilliant Blue R-250 and destained with 40 methanol and 10 acetic acid. Another gel was analyzed by
2D Western blot.Apoptosis Analysis by Flow Cytometry AssayThe cells (36105 per well) were plated in 6-well plates and treated with NS, control IgG (200 mg/mL) or PAb (200 mg/mL). After 48 h, flow cytometric analysis was conducted to identify subG1 phase cells/apoptotic cells. Briefly, the cells were suspended in 1 mL of hypotonic fluorochrome solution containing 50 mg/mL propidium iodide in 0.1 sodium citrate with 0.1 Triton X-100 and analyzed by a flow cytometer. Apoptotic cells appeared in the cell cycle distribution as cells with DNA content less than that of G1 phase cells.Antitumor Effect of PAb on Xenograft SCID Mouse Models with ARH-Human ARH-77 MM cells (56106) were.
