Detection of CD3ζ and CD62L in CTL by real-time PCR and Western-blot
CTL, CTL+MDSC(1:4) and CTL+MDSC(1:4)+DOX(2µmol/L) were incubated respectively. The contents of CD3ζ and CD62L in CTL of the different groups were assessed and compared by real-time PCR. Total RNA in samples was isolated using the ultra-pure RNA extraction kit according to the manufacturer’s guidelines (Cwbio, Co. Ltd.). For reverse transcription, 1 μg of RNA was used to synthesize single-strand cDNA (HiFi-MMLV cDNA first strand synthesis kit; Cwbio, Co. Ltd.) according to the manufacturer’s guidelines. Real-time PCR was performed using a fluorescence quantitative PCR amplifier (LightCycler 480 II, Roche Inc., Germany), as described elsewhere. Triplicate reactions were set up for each gene in a 96-well plate. Reaction information is summarized in Table 1. Amplification was performed with the following program: initial denaturation at 95°C for 15 min, followed by 40 cycles of 95°C for 10 s, 58°C for 30 s and 72°C for 30 s. The experimental data were expressed as relative expression using the 2-ΔΔCt equation as described previously 15.
Proteins were extracted with RIPA lysis buffer (Invitrogen) and quantitatively detected with the BCA Protein Assay kit (Invitrogen). The samples were separated by 12% SDS-PAGE, transferred to a PVDF membrane, and blocked by 5% BSA. The primary antibodies against each protein were added and incubated at 4°C overnight. Afterward, the second antibodies were added and incubated for 4 h at 20°C. After washing the membrane, chemiluminescence was detected on X-ray film by FluorChem®HD2 (Alpha Innotech, Santa Clara, CA, USA), with GAPDH as the internal reference.