HEp-2 (catalogue #CCL-23) and HEK293 (CRL-1573) cells were obtained from American Type Cell Collection (Manassas, VA) and maintained in DMEM supplemented with 10% fetal calf serum, L-glutamine (2 mM), penicillin (200 U/ml), and streptomycin (200 ug/ml). Cells were transfected using the Effectene transfection system (Qiagen, Valencia, CA) as directed by the manufacturer. HEp-2 cells were used for immunohistochemistry; twenty-four hours after transfection, cells were fixed with 2% paraformaldehyde in PBS and permeabilized with 0.2% triton X-100 in PBS. Cells were stained with primary and secondary antisera as indicated.
To investigate the interaction between NLS-EDC4 and PATL1, cells were transfected with both plasmids and subsequently treated for 2 hours with leptomycin B (LMB; 10nM) to inhibit nuclear export.
To deplete endogenous P-bodies from HEp-2 cells, cells were transfected with siRNA directed against LSm14a, DDX6, or with scrambled siRNA using Dharmafect (Dharmacon, Lafayette, CO) as directed by the manufacturer. Twenty-four hours after treatment with siRNA, cells were transfected with plasmids encoding P-body components. Cells were subsequently fixed, permeabilized and stained with primary and secondary antisera.
To permit semi-quantitative analysis of the interaction between P-body components in the two-hybrid assay, the number of transfected cells expressing two (or more) P-body components in nuclear dots was compared to the total number of cells expressing two (or more) of the transfected proteins. Three separate transfections were performed and at least 30 cells expressing two (or more) proteins were scored in each transfection. Results are presented as mean +/- SEM.
For co-immunoprecipitation assays, HEK293 cells were transfected with plasmids encoding GFP-EDC4 and monomeric cherry (mCh) fused to XRN1(1232–1706), GFP-EDC4(630–1437 and mCh-XRN1(1232–1706) or with mCh-XRN1(1232–1706) alone. Twenty-four hours after transfection, cells were incubated with lysis buffer containing Tris-HCl pH7.4 (25 mM), NaCl (150 mM), EDTA (1mM), NP-40 (1%) and glycerol (5%). The GFP-containing fusion proteins were immunoprecipitated using GFP-Trap magnetic agarose, as directed by the manufacturer (Proteintech, Rosemont, Illinois). Proteins were eluted from the magnetic agarose by boiling in 2X sample buffer.
To investigate the interaction between NLS-EDC4 and PATL1, cells were transfected with both plasmids and subsequently treated for 2 hours with leptomycin B (LMB; 10nM) to inhibit nuclear export.
To deplete endogenous P-bodies from HEp-2 cells, cells were transfected with siRNA directed against LSm14a, DDX6, or with scrambled siRNA using Dharmafect (Dharmacon, Lafayette, CO) as directed by the manufacturer. Twenty-four hours after treatment with siRNA, cells were transfected with plasmids encoding P-body components. Cells were subsequently fixed, permeabilized and stained with primary and secondary antisera.
To permit semi-quantitative analysis of the interaction between P-body components in the two-hybrid assay, the number of transfected cells expressing two (or more) P-body components in nuclear dots was compared to the total number of cells expressing two (or more) of the transfected proteins. Three separate transfections were performed and at least 30 cells expressing two (or more) proteins were scored in each transfection. Results are presented as mean +/- SEM.
For co-immunoprecipitation assays, HEK293 cells were transfected with plasmids encoding GFP-EDC4 and monomeric cherry (mCh) fused to XRN1(1232–1706), GFP-EDC4(630–1437 and mCh-XRN1(1232–1706) or with mCh-XRN1(1232–1706) alone. Twenty-four hours after transfection, cells were incubated with lysis buffer containing Tris-HCl pH7.4 (25 mM), NaCl (150 mM), EDTA (1mM), NP-40 (1%) and glycerol (5%). The GFP-containing fusion proteins were immunoprecipitated using GFP-Trap magnetic agarose, as directed by the manufacturer (Proteintech, Rosemont, Illinois). Proteins were eluted from the magnetic agarose by boiling in 2X sample buffer.
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