On targetplus smartpool rat tmed10 sirna

Tmp21 was depleted by two sequential knockdowns using ON-TARGETplus SMARTpool Rat Tmed10 siRNA (Dharmacon L-092937-01-0005) and Dharmafect 2 transfection reagent (Dharmacon T-2002-02) exactly as recommended by the manufacturer. The second knockdown was performed 48 hr after the first, and experiments were performed 24 hr after the second knock down.

All experiments were performed in stably transfected normal rat kidney (NRK) cells, unless specified. All cells cotransfected with organelle markers were imaged 48 hr after transient transfection. YFP-PrP was constructed by replacing the endogenous PrP signal sequence (residues 1–22) with the bovine preprolactin-signal sequence to ensure efficient translocation into the ER (Rane et al., 2004 (link)) and inserting mYFP into the unique Bsu36I site. Construction of eGFP-CD59, GFP-Thy1, GFP-DAF, and GFP-FolR is described in the Extended Experimental Procedures. Misfolding mutants were derived by site-directed mutagenesis. Tmp21 was depleted by two sequential knockdowns within 48 hr using 50 nM concentration of ON-TARGETplus SMARTpool Rat Tmed10 siRNA and Dharmafect 2 transfection reagent (Dharmacon), and experiments were performed 24 hr after the second knockdown.

Tmp21 was depleted by two sequential transfections 48 h apart of the ON-TARGETplus SMARTpool Rat Tmed10 siRNA (L-092937-01-0005; Dharmacon) with Dharmafect 2 transfection reagent (T-2002-02; Dharmacon) exactly as recommended by the manufacturer. Immediately after the second siRNA transfection, cells were transferred to coverslips, and 24 h later, the imaging experiments were performed.