Journal:

Article Title: Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

doi: 10.1016/j.taap.2009.12.035

Figure Lengend Snippet: (A) Quantification of mouse Mrp2 in the membrane vesicles prepared from the mPCT-A2 cells (mPCT-A2) transiently expressing mouse Mrp2 (Ex) and from control untransfected (Un) and mock-transfected (Mock) mPCT-A2 cells were resolved on 7.5% SDS-PAGE, transferred onto PDF membranes and probed with RP-4 mouse Mrp2 specific antibody (IgG) and with the same antibody preincubated with the peptide (10:1) used for immunization (IgG + P). Loading: 7 μg. mPCT cells (mPCT) endogenously express Mrp2. The membranes were isolated from mPCT cells using the technique described for mPCT-A2 cell membrane isolation, resolved on 7.5% SDS-PAGE and immunoblotted with RP-4 antibody (IgG) and with the same antibody preincubated with the peptide used for immunization (IgG + P). Loading: 50 μg.

Article Snippet: To suppress Mrp2 synthesis, the cells were transiently transfected with the anti-mouse Mrp2 SureSilencingTM (shRNA) plasmid (SuperArray Biosciences, Frederick, MD) using the Lipofectamine method (Invitrogen).

Techniques: Membrane, Expressing, Control, Transfection, SDS Page, Isolation

Journal:

Article Title: Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

doi: 10.1016/j.taap.2009.12.035

Figure Lengend Snippet: (A) The ATP-dependent transport of Ac-DCVC (black bars) and DCVC (grey bars) into membrane vesicles prepared from the mPCT cells expressing mouse Mrp2 (Ex) measured by LC/MS/MS. ATP was replaced with an equimolar concentration of AMP in control experiments (white bars). * Significant difference (p<0.05) from control with AMP instead of ATP. (B) A representative curve of ATP-dependent Ac-DCVC transport inside Mrp2 vesicles. The data are means ± S.D. of two measurements. (C) The Lineweaver-Burk plot of the data shown in (B). S0.5 values were determined as the substrate concentration at half-maximal velocity of transport under the experimental conditions described above using the double-reciprocal plot (Lineweaver, Burk, 1934).

Article Snippet: To suppress Mrp2 synthesis, the cells were transiently transfected with the anti-mouse Mrp2 SureSilencingTM (shRNA) plasmid (SuperArray Biosciences, Frederick, MD) using the Lipofectamine method (Invitrogen).

Techniques: Membrane, Expressing, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Control

Journal:

Article Title: Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

doi: 10.1016/j.taap.2009.12.035

Figure Lengend Snippet: (A) Apical to basolateral and (B) basolateral to apical transport of Ac-DCVC through the monolayer of mPCT cells (white bars) or the mPCT cells transfected with anti-mouse Mrp2 shRNA plasmid (black bars). In control experiments (C) mPCT cells were transfected with a nonspecific anti-AA3 shRNA plasmid (Fig. 3C). Note that mPCT cells do not express AA3 (Newman et al., 2007). (D) Anti-Mrp2 shRNA significantly decreased the expression of Mrp2 protein in mPCT cells in comparison with untransfected cells (control) whereas anti-AA3 shRNA did not. A representative immunoblot is shown on the left. The membranes were isolated from untransfected (Control) and transfected with AA3 and Mrp2 shRNA plasmids mPCT cells using the technique described for mPCT-A2 cell membrane isolation, resolved on 7.5% SDS-PAGE and immunoblotted with RP-4 antibody. Loading: 50 μg. Graphic illustration of Mrp2 quantification in 3 separate experiments is shown on the right. The bands were scanned and quantified in Adobe Photoshop (Adobe Instruments). * Shows significant difference (p<0.05) between transfected and untransfected (control) cells. (E) Apical to basolateral and basolateral to apical transport of DCVC through the monolayer of mPCT cells (white bars) and the mPCT cells transfected with anti-mouse Mrp2 shRNA plasmid (black bars). * Shows significant difference (p<0.05) between transfected and untransfected (control) cells.

Article Snippet: To suppress Mrp2 synthesis, the cells were transiently transfected with the anti-mouse Mrp2 SureSilencingTM (shRNA) plasmid (SuperArray Biosciences, Frederick, MD) using the Lipofectamine method (Invitrogen).

Techniques: Transfection, shRNA, Plasmid Preparation, Control, Expressing, Comparison, Western Blot, Isolation, Membrane, SDS Page