The largest database of trusted experimental protocols

3 protocols using perk inhibitor gsk2606414

1

Antibody Reagents for Heparanase Research

Check if the same lab product or an alternative is used in the 5 most similar protocols
Anti-heparanase polyclonal antibody was purchased from Prospec (Rehovot, Israel). Anti-Hpa2 polyclonal (Ab 58) and monoclonal (20c5) antibodies have been described previously [4 (link)]. Anti-GRP78 (Bip), anti-CHOP and anti-ATF3 (ab254268) antibodies were purchased from Abcam (Cambridge, UK). Anti-AFT4 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz CA); Anti-HIF1-α antibody (610958) was purchased from BD Transduction Laboratories. Anti-ATF3 polyclonal antibody has been described previously [28 (link)]. Anti-actin antibody, thapsigargin, tunicamycin, MG132, Cisplatin, PERK inhibitor GSK2606414, and L-leucyl-L-leucine methyl ester (LLOMe) were purchased from Sigma. Tissue arrays of cervical and hepatocellular carcinomas were purchased from US Biomax (Rockville, MD).
+ Open protocol
+ Expand
2

HaCaT cell culture and CMIT treatment

Check if the same lab product or an alternative is used in the 5 most similar protocols
Human keratinocyte HaCaT cells (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle medium (WELGENE, Gyeongsangbuk-do, Korea) supplemented with 10% heat-inactivated fetal bovine serum (WELGENE) and 50 U/mL penicillin and 50 μg/mL streptomycin (WELGENE) at 37°C in a humidified atmosphere containing 5% CO2. CMIT (also known as 5-Chloro-2-methyl-4-isothiazolin-3-one; CAS No. 26172-55-4, purity > 65%) was purchased from Key Organics-Bionet Research (Camelford, UK). PERK inhibitor (GSK2606414) and sodium arsenite were purchased from Sigma-Aldrich (St. Louis, MO, USA).
+ Open protocol
+ Expand
3

Induction of ER Stress and ISR Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols
To induce ER stress, cells were treated with 5 μg/ml tunicamycin (TM; Sigma‐Aldrich) or 1 μM thapsigargin (TG; Santa Cruz Biotechnology) for indicated times. HRI activation was triggered by 5 μM sodium arsenite (Sigma‐Aldrich) treatment for 20 h, PKR activation by 20 μM BEPP (Sigma‐Aldrich) treatment for 6 h and GCN2 activation by 10 nM Halofuginone (Cayman Chemical Company) treatment for 20 h, and ROS was generated by 100 μM menadione (Men; Sigma‐Aldrich) treatment for 4 h. Treatment with 0.5 μM PERK inhibitor GSK2606414 (Sigma‐Aldrich), 40 μM IRE1α inhibitor 4μ8C (Tocris), 75 μM angiogenin inhibitor NSC‐65828 [8‐amino‐5‐(4′‐hydroxybiphenyl‐4ylazo)naphthalene‐2‐sulfonate] (National Cancer Institute (NCI)) and 5 μM mTOR inhibitor Torin1 (Tocris) was started 1 h before induction of ER stress. Treatment with 200 nM ISR inhibitor ISRIB (Selleck Chemicals) was started 2 h before fixation, and treatment with 5 μM HRI inhibitor hemin chloride (Santa Cruz Biotechnology) was started 3 h before fixation. Compounds were dissolved in H2O (sodium arsenite), 100 mM NaOH (hemin chloride) or DMSO (rest). For all treatments, equal volumes of solvents were added to the controls. To determine ROS levels, cells were incubated with 5 μM CellROX green reagent (Thermo Fisher) 30 min before fixation.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!