Steponeplus real system

J774.1 cells at 1 × 10⁵ were seeded into 6-well plates and allowed to adhere overnight. Cells were stimulated with lipopolysaccharide (10 ng/ml) for 6 h in the presences or absence of guanabenz (50 μM) as previously reported (23 (link)). Total RNA was isolated from the cells using TRIzol LS (Invitrogen), and cDNA was generated using Omniscript (Qiagen). RT-qPCR was carried out using SYBR Green real-time PCR master mixes (Invitrogen) and the StepOnePlus Real system (Applied Biosystems). Relative levels of transcripts were calculated with the ΔΔC_T method using GAPDH as the internal control. The relative levels of the target mRNAs from the untreated cells were adjusted to 1 and served as the basal control value. Each experiment was performed three times, each with three technical replicates. The primers used are listed in Table S1 in the supplemental material.

RNA was isolated from parasites using TRIzol LS (Invitrogen), and cDNA was generated using Omniscript (Qiagen). RT-qPCR was carried out using specific primers (Table S1) with SYBR green real-time PCR master mixes (Invitrogen) and the StepOnePlus Real system (Applied Biosystems). Relative levels of transcripts were calculated with the ΔΔC_T method using the GAPDH gene as an internal control. Each experiment was performed three times, each with three technical replicates.