Validation of MYD88 Total assay in THP-1 derived macrophages and Karpas299 cells

THP-1 cells were seeded in a 96-well culture plate (80,000 cells/well) in complete medium, treated with 100 nM PMA and incubated for 24 hours at 37°C, 5% CO2. The cells were further treated with increasing concentrations of LPS for 24 hours. After treatment, the cells were washed in HBSS and lysed with 100 µL of lysis buffer for 10 minutes at RT with shaking at 350 rpm. Lysates were further diluted 1:5 in lysis buffer before proceeding to the detection step. MYD88 Total and NFkB Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 1,600 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision Nexus™ using standard AlphaLISA settings.

Karpas299 cells were harvested, washed in HBSS + 0.1% BSA and seeded in a 96-well culture plate (100,000 cells/well) in HBSS + 0.1% BSA. The cells were further treated with increasing concentrations of IL-1b for 10 minutes. 

After treatment, the cells were lysed with 50 µL of 5X lysis buffer for 10 minutes at RT with shaking at 350 rpm. MYD88 Total, IRAK4 Phospho (Thr345/Ser346) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision Nexus using standard AlphaLISA settings.

Specificity of MYD88 Total assay

Total MYD88 protein levels were assessed in A549 wild type (WT) cells and MYD88 knockout (KO) A549 cells. Both cell lines were grown to confluency in a T175 flask in complete medium. Each flask was lysed in 4 mL of lysis buffer for 10 minutes at RT with shaking at 350 rpm. Lysates were serially diluted in lysis buffer and evaluated for MYD88 Total as well as Cofilin Total levels to ensure normalization (data not shown). For the detection step, 10 µL of cell lysate were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision Nexus using standard AlphaLISA settings. As expected, MYD88 was detected in the WT-A549 cells but not in the MYD88-KO cell line.

HeLa cells were seeded at different densities in a 96-well plate (200 µL/well) in complete culture medium and incubated overnight at 37°C, 5% CO2. Cells were siRNA treated with 50 µL of a mix of transfection reagent (Santa Cruz, sc-29528)/MYD88 siRNA (Santa Cruz, sc- 35986) or a mix of transfection reagent/siRNA control (Santa Cruz, sc-37007) for 48 hours. After treatment, the cells were lysed with 100 µL of lysis buffer for 10 minutes at RT with shaking at 350 rpm. MYD88 Total levels were evaluated by AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision Nexus using standard AlphaLISA settings.