All animal experiments were approved by the Animal Care and Use Committee of the Yonsei University College of Medicine. Male C57BL/6J mice (B6, [SLC-M-0133]) aged 8 weeks were used in the animal experiments, which is purchased from Japan SLC, Inc. (Hamamatsu, Japan). All mice had free access to water and food and were housed in cages maintained at 23 ± 2 °C under a 12 h light/12 h dark cycle and 50%–70% humidity. To the depletion of PERK in mice, PERK siRNA (20 nM, Santa Cruz Biotechnology; sc-36214) was injected through the tail vein along with the Invivofectamine 3.0 reagent (Thermo Fisher Scientific, IVF3001) for 3 days according to the manufacturer's instructions. Mice were fed a normal chow diet (LabDiet, 5053) without fasting or a high-carbohydrate diet (HCD; Dyets, 102235) after 24 h of fasting. After 18 h of refeeding, the mice were sacrificed. The serum alanine aminotransferase (ALT) levels were measured via colorimetric determination using an activity assay kit (FUJIFILM, 3250).
Perk sirna
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany
PERK siRNA is a small interfering RNA (siRNA) designed to target the PERK gene, which encodes the Protein Kinase R (PKR)-like Endoplasmic Reticulum Kinase. The core function of PERK siRNA is to induce the specific knockdown of PERK expression in cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Atf4 sirna, by Santa Cruz Biotechnology (2 mentions)
Invivofectamine 3.0 reagent, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Sirna transfection reagent, by Santa Cruz Biotechnology (1 mentions)
Prk atf4, by Addgene (1 mentions)
Scrambled control sirna, by Santa Cruz Biotechnology (1 mentions)
Sesn2 sirna, by Santa Cruz Biotechnology (1 mentions)
Caspase 12, by Santa Cruz Biotechnology (1 mentions)
Mcp 1 elisa kit, by R&D Systems (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Atg5 sirna, by Cell Signaling Technology (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
6 protocols using perk sirna
1
Depletion of PERK Modulates Hepatic Metabolism
2
MCF7 Cell Transfection and Citreoviridin Treatment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MCF7 cells (1 × 105) were incubated in six-well culture plates for 24 h before transfection. The confluency of the cells at the time of transfection was 30–50%. Cells were transfected with 80 pmol of the control or with PERK siRNA (Santa Cruz Biotechnology) with 7.5 μl Lipofectamine 2000 (Invitrogen) in serum-free DMEM according to the manufacturer's instructions. After transfection for 4 h at 37 °C, the medium was replaced with DMEM containing 10% FBS. After 36 h of transfection, cells were treated with 0.1% DMSO or with 0.1 μM citreoviridin. Cells were collected after 24 h of treatment and then stored at −80 °C until protein analysis.
3
Transient GRP78 and PERK Knockdown Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To develop cells with transient GRP78 knockdown, Caki-1 and UMRC-3 cells were transiently transfected with a 19-bp small interfering RNA (siRNA) targeting GRP78 (sense: 5′-GGAGCGCAUUGAUACUAGA-3′, anti-sense: 5′-GCCUAGGUCUCUUAGAUGA-3′) or a non-silencing siRNA (si-Scr) [31 (link)]. Caki-1 and UMRC-3 cells were seeded onto 6-well plates at a density of 1.2 × 105 cells/well. At 30%–50% confluence, cells were transfected for 16 h with the siRNAs using Lipofectamine 2000 (Invitrogen Life Technologies) diluted with OPTIMEM (Invitrogen Life Technologies) according to the manufacturer's instructions. After transfection, the medium was replaced with fresh medium, and cells were incubated for 48–72 h according to the purpose of the experiment. To develop cells with transient protein kinase-like ER kinase (PERK) knockdown, GRP78-overexpressing Caki-1 and parental Caki-1 cells were transfected with PERK siRNA (Santa Cruz Biotechnology).
4
Modulation of ATF4 and CEBPB in MDA-MB-231 Cells
5
Endoplasmic Reticulum Stress and Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EP, thapsigargin (THA, an ERS inducer), tumor necrosis factor-α (TNF-α), dimethyl sulfoxide (DMSO) and the antibody against TNF-α were purchased from the Sigma-Aldrich Company (St. Louis, MO, USA). The Cell Counting Kit-8 (CCK8) was purchased from Dojindo (Kumamoto, Japan). PERK siRNA and the antibodies against GRP78, intercellular cell adhesion molecule 1 (ICAM-1), matrix metalloproteinase 9 (MMP9) and caspase12 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human soluble adhesion molecule ICAM-1 (sICAM-1), soluble E-selectin (sE-selectin), interleukin (IL-8) and monocyte chemoattractant protein-1 (MCP-1) ELISA kits were obtained from R&D Systems (Minneapolis, MN, USA). The antibodies against phosphorylated-PERK (p-PERK), PERK, ATF4 and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA). The rabbit anti-goat, goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from the Zhongshan Company (Beijing, China).
6
Autophagy Regulation in LPS-Treated A549 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were transiently transfected with an enhanced green fluorescent protein (EGFP)-LC3 plasmid (constructed by GeneChem Co. Ltd, Shanghai, China) using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer's instructions. After a 48-h transfection, the cells were treated with LPS at 50 µg/ml for another 16 h and then subjected to laser confocal scanning microscopy (TCS SP5; Leica Microsystems, Mannheim, Germany).
For the RNA interference experiments, A549 cells were transfected with Beclin-1 short interfering RNA (siRNA), Atg5 siRNA (Cell Signaling), PERK siRNA, ATF4 siRNA (Santa Cruz), or control siRNA or co-transfected with EGFP-LC3 plasmid using Lipofectamine 3000 Transfection Reagent (Invitrogen) as directed by the manufacturer. After 48 h, the effect of the siRNA treatment was assessed via Western blotting analysis. In parallel, cells were treated with LPS and analysed as described above.
For the RNA interference experiments, A549 cells were transfected with Beclin-1 short interfering RNA (siRNA), Atg5 siRNA (Cell Signaling), PERK siRNA, ATF4 siRNA (Santa Cruz), or control siRNA or co-transfected with EGFP-LC3 plasmid using Lipofectamine 3000 Transfection Reagent (Invitrogen) as directed by the manufacturer. After 48 h, the effect of the siRNA treatment was assessed via Western blotting analysis. In parallel, cells were treated with LPS and analysed as described above.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!