RAW264.7 and TLR4-deficient (3 × 106) macrophage cells were grown in culture plates. After 4 hrs of growth, cells were stimulated with proteins. Post-treatment completion, cells were rinsed with 1× PBS (RT) twice and treated with 2× SDS dye [25 (link)]. The protein was run in varying percentages of SDS-PAGE gel depending on the size of the proteins to be probed. The protein samples were transferred to the blotting membrane (PVDF) for western blot analysis. The membranes were then probed using the primary antibodies against the respective antigens, such as NFKB1, pP-65, cytochrome C (CytC), LC3BII, SQSTM1, beclin1, pMTOR, pULK1, pAKT, pPI3K, HSP60, pcFOS, tcFOS, pJNK1/2, tJNK1/2, tERK1/2, pERK1/2, tP38, pP38, pyruvate dehydrogenase (PD), succinate dehydrogenase (SDHA), CoxIV and Rab7, GAPDH and β-actin antibodies (Cell Signalling). The location of MoxR1 in various purified cellular materials of M. tb was investigated using the antibody against MoxR1 (αMoxR1) (BEI, Resources, NIAID, NIH, USA). The affinity-purified His-tagged MoxR1 protein was confirmed using a monoclonal mouse anti-His antibody (Sigma, USA). For signal generation, secondary antibodies were used as appropriate. Rapamycin and bafilomycin A1 treatment are used for 6 hrs along with MoxR1 treatment to explore its function in autophagy regulation. The protein samples were then prepared by lysis and processed [7 (link),26 (link)]. The western blotting performed to detect the proteins onto the PVDF membrane. 5% BSA in TBST was used for blocking the PVDF membrane in the western blotting of phosphoproteins. The band intensities of proteins were quantified using ImageJ software. GAPDH, β-actin, tFos, tERK1/2, tP38, or tJNK1/2 was used to normalize protein bands as required.
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