HTRF vs WB using total EIF2a assay on human HeLa cells

HeLa cells were grown in a T175 cm² flask for 2 days in MEM alpha medium supplemented with 10% FBS. The cells were treated for 10 minutes with 100 nM Calyculin A at 37°C before lysis with 3 mL of 1X supplemented lysis buffer #4. The cell lysate was serially diluted in the supplemented lysis buffer #4 and 16 µL of each dilution were analyzed in parallel by HTRF or by Western Blot. Using the HTRF Total EIF2 alpha kit, only 0.2 µg of total proteins were sufficient for minimal signal detection, demonstrating that the HTRF assay is 4 fold more sensitive than the Western Blot.

 

Pharmaco effects of Febrifugine on Total and Phospho (Ser52) EIF2a

HepG2 and NIH-3T3 cells were treated with increasing concentrations of Febrifugine. After lysis, lysates were transferred over into a low volume white microplate before adding 4 µL of the HTRF phospho EIF2 alpha or total EIF2 alpha detection antibodies. The HTRF signal was recorded after an overnight incubation. Febrifugine treatment induces a good increase in EIF2 alpha phosphorylation on Ser52, while its expression level remains stable.

 

 

Pharmaco effects of Thapsigargin on Total and Phospho (Ser52) EIF2a

Dose response experiments were performed on HepG2 cells with Thapsigargin, an inhibitor of the sarco/endoplasmic reticulum Ca2+/ATPase by inducing ER stress. Using the two plate protocol, cells were treated with increasing concentrations of Thapsigargin and lysed. Lysates were transferred twice over into a low volume white microplate before finally adding HTRF phospho-EIF2 alpha or total EIF2 alpha detection antibodies. Results showed EIF2 alpha expression level remains stable, demonstrating that there is no cytotoxic effect of the compound on cells.

 

Pharmaco effects of GSK2606414 on Total and Phospho (Ser52) EIF2a

HepG2 and C2C12 cells were cultured and treated with increasing concentrations of GSK2606414, a selective PERK inhibitor, before activation with 500 nM Thapsigargin. After medium removal, the cells were lysed and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF phospho EIF2 alpha or total EIF2 alpha detection antibodies. The HTRF signal was recorded after an overnight incubation.

 

 

Pharmaco effects of Palmitate on Total and Phospho (Ser52) EIF2a

Dose response experiments were performed on HepG2 cells with a solution of palmitate conjugated to BSA using the two plate assay protocol. Cells were then treated with increasing concentrations of palmitate/BSA for 6 hours at 37°C - 5% CO2. After lysis buffer, 16 µL of lysate were transferred twice over into a low volume white microplate before finally adding phospho EIF2 alpha or total EIF2 alpha detection antibodies. The HTRF signal was recorded and by way of a 2-way ANOVA test, results demonstrated Palmitate induces a good, significant increase in the phosphorylation of EIF2 alpha on SER52.