siRNA(h) ATG5

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 20 min at RT and add to cells. Replace medium after 4-6h with 10% heat-inactivated FBS and re-incubate cells at 37°C.

Protocol tips
Add diluted siRNA to diluted Lipofectamine Reagent Incubate for 20 min at RT and add to cells. Replace medium after 4-6h with 10% heat-inactivated FBS and re-incubate cells at 37°C.

Publication protocol

Small-interfering RNA (siRNA) against Atg5 and a non-specific scrambled siRNA were purchased from Ambion. All siRNAs were synthesized by Qiagen (Chatsworth, CA, USA). HepG2 cells were cultured in 6-well plates. Lipofectamine 2000 (Invitrogen, Rockville, MD, USA) was mixed into serum-free DMEM containing siRNA1, siRNA2 or scrambled RNA. Additionally, mock controls were transfected with Lipofectamine 2000 alone, incubated at room temperature for 20 min and distributed into duplicate wells. Transfections were performed at 37°C in 5% CO2. Media were replaced with DMEM containing 10% heat-inactivated FBS for 4–6 h. After 2 h, cells were treated as indicated.

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Manufacturer protocol

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