ON-TARGETplus Mouse Rheb (19744) siRNA - SMARTpool

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	RAW 264.7 cells, a murine macrophage cell line, were acquired from ATCC (Manassas, VA) and were maintained in Dulbecco ’ s Modifi ed Eagle ’ s medium (GIBCO BRL, Grand Island, NY) containing 20 m MHEPES (Fisher scientifi c, Atlanta, GA), 10% FBS (GIBCO BRL), 100 U/ml of penicillin and 100 μg/ml of streptomycin (Bio Whittaker Inc., Walkersville, MD) at 37 ° C in 5% CO 2. The cells were plated onto 6- or 96-well plates.For silencing Beclin 1, BNIP3, Rheb and Atg5 genes, Beclin 1-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-055895-00-0005), BNIP3-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-040256-01-0005) and Rhebdirected siRNA pool (ON-TARGET plus SMARTpool reagent L-057044-00-0005) were purchased from Dharmacon (Lafayette, CO). Atg5-directed siRNA was purchased from Ambion (Austin, TX). The sequence of Atg5 siRNA is 5 ’ -ACC GGA AAC TCA TGG AAT A-3 ’ . Negative control siRNA was purchased from Dharmacon. Cells were transfected with Beclin 1, BNIP3, Rheb, Atg5 or control siRNA by electroporation under conditions of 1350 voltage and 35 ms using a pipettetype electroporator (MicroPorator-Mini, Digital Bio Technology, Suwon, Kyounggi-do, Korea) according to the manufacturer ’ s instructions. The effi ciency of the siRNA silencing of the designated gene was confi rmed by western blot analysis at 48 h after transfection

Protocol tips

RAW 264.7 cells, a murine macrophage cell line, were acquired from ATCC (Manassas, VA) and were maintained in Dulbecco ’ s Modifi ed Eagle ’ s medium (GIBCO BRL, Grand Island, NY) containing 20 m MHEPES (Fisher scientifi c, Atlanta, GA), 10% FBS (GIBCO BRL), 100 U/ml of penicillin and 100 μg/ml of streptomycin (Bio Whittaker Inc., Walkersville, MD) at 37 ° C in 5% CO 2. The cells were plated onto 6- or 96-well plates.For silencing Beclin 1, BNIP3, Rheb and Atg5 genes, Beclin 1-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-055895-00-0005), BNIP3-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-040256-01-0005) and Rhebdirected siRNA pool (ON-TARGET plus SMARTpool reagent L-057044-00-0005) were purchased from Dharmacon (Lafayette, CO). Atg5-directed siRNA was purchased from Ambion (Austin, TX). The sequence of Atg5 siRNA is 5 ’ -ACC GGA AAC TCA TGG AAT A-3 ’ . Negative control siRNA was purchased from Dharmacon. Cells were transfected with Beclin 1, BNIP3, Rheb, Atg5 or control siRNA by electroporation under conditions of 1350 voltage and 35 ms using a pipettetype electroporator (MicroPorator-Mini, Digital Bio Technology, Suwon, Kyounggi-do, Korea) according to the manufacturer ’ s instructions. The effi ciency of the siRNA silencing of the designated gene was confi rmed by western blot analysis at 48 h after transfection

Publication protocol

RAW 264.7 cells, a murine macrophage cell line, were acquired from ATCC (Manassas, VA) and were maintained in Dulbecco ’ s Modifi ed Eagle ’ s medium (GIBCO BRL, Grand Island, NY) containing 20 m MHEPES (Fisher scientifi c, Atlanta, GA), 10% FBS (GIBCO BRL), 100 U/ml of penicillin and 100 μg/ml of streptomycin (Bio Whittaker Inc., Walkersville, MD) at 37 ° C in 5% CO 2. The cells were plated onto 6- or 96-well plates.For silencing Beclin 1, BNIP3, Rheb and Atg5 genes, Beclin 1-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-055895-00-0005), BNIP3-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-040256-01-0005) and Rhebdirected siRNA pool (ON-TARGET plus SMARTpool reagent L-057044-00-0005) were purchased from Dharmacon (Lafayette, CO). Atg5-directed siRNA was purchased from Ambion (Austin, TX). The sequence of Atg5 siRNA is 5 ’ -ACC GGA AAC TCA TGG AAT A-3 ’ . Negative control siRNA was purchased from Dharmacon. Cells were transfected with Beclin 1, BNIP3, Rheb, Atg5 or control siRNA by electroporation under conditions of 1350 voltage and 35 ms using a pipettetype electroporator (MicroPorator-Mini, Digital Bio Technology, Suwon, Kyounggi-do, Korea) according to the manufacturer ’ s instructions. The effi ciency of the siRNA silencing of the designated gene was confi rmed by western blot analysis at 48 h after transfection

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