Alliance mini 4m

Whole cells were lysed in RIPA buffer (ELPIS biotech, Daejeon, Korea) with a protease inhibitor cocktail. Extracted proteins were resolved by 12% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Then proteins were blocked with TBS-T containing 5% skim milk (Santa Cruz Biotechnology) at room temperature for 1 h and incubated overnight at 4 °C with the primary antibody (1:1000). The PVDF membrane was washed in TBS-T and incubated for 2 h at room temperature with HRP-conjugated secondary antibody (1:2000). β-actin was used loading control for protein. The bound antibodies were detected using chemiluminescent HRP substrate (ECL, Millipore, Billerica, MA). The blots were exposed to Fuji medical X-Ray film RX-N (Fuji, Japan) or quantified using an Alliance Mini 4M (UVITEC Cambridge, UK).

Total RNA was isolated from Mtb-infected BMDMs and mRNA was reverse transcribed into cDNA. Reverse transcription PCR was performed using Prime Taq Premix (Genet Bio, Daejeon, South Korea) to detect the mRNA levels of target genes. For quantitative real-time PCR, cDNA was synthesised and target gene expression was quantified by SYBR green (QIAGEN, Venlo, The Netherlands). The mean value of triplicate reactions was normalised against the mean value of β-actin.
For Western blotting, Mtb-infected cells were lysed in radioimmunoprecipitation (RIPA) assay buffer containing protease inhibitor cocktail (PIC), and whole-cell lysates were separated on a 12% SDS-PAGE gel before being transferred to a polyvinylidene difluoride (PVDF) membrane. Primary antibodies against SLC7A2 (Abcam, Cambridge, UK) were diluted 1:1,000 overnight at 4 °C, and goat anti-rabbit IgG (Santa Cruz, Dallas, TX, USA) was used as a horseradish peroxidase (HRP)-conjugated secondary antibody and was diluted 1:2,000 for 2 h at room temperature. β-actin (1:5,000, Santa Cruz) was used as a loading control. To detect the target proteins, membranes were developed using a chemiluminescent HRP substrate reagent (ECL, Millipore, Billerica, MA, USA) and subsequently quantified using the Alliance Mini 4 M (UVitec, Cambridge, UK).