Figure 4.

M. tb PE6 targeted to nucleus/nucleolus and mitochondria in transiently transfected HEK293T cells interacts with Nucleolin. (A, B) Confocal laser scanning microscopic analysis of GFP and GFP-PE6 expressing HEK293T cells. Transiently transfected cells were fixed using 4% formaldehyde and washed with cold PBS. GFP-PE6 (green) and GFP (green) localization was analyzed at 24, 48, and 72 h post-transfection. Nucleolus was stained using an anti-Nucleolin antibody (pseudo red). Mitotracker deep red FM was used to study the mitochondrial localization (pseudo yellow). DAPI was used to stain the nucleus (blue). A547 conjugated secondary antibody was used for signal generation. Scale bars indicate 10 µm. (C) Western blots showing the Nucleolin level in macrophage cells infected with recombinant M. smegmatis containing pe6 and vector. After 24 h of infection, cell lysate was prepared and fractionated on SDS-PAGE, and proteins were transferred onto the PVDF membrane. Nucleolin levels were analyzed using the anti-Nucleolin antibody. Untreated and vector alone transformed cells were used as controls. β-actin was used as a loading control. (D) Densitometric quantitation of Nucleolin bands was represented as a bar graph. Nucleolin level was normalized to respective β-actin bands and represented as [%] protein levels to β-actin. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison post-test. Data are representative of three independent experiments. ***P < 0.001 vs. control. (E, F) Western blots showing Co-IP of PE6 and Nucleolin from cell extracts prepared from HEK293T cells expressing GFP and GFP-PE6 using anti-PE6 and anti-Nucleolin antibodies, respectively. Lysates prepared from HEK293T cells expressing GFP were used as a negative control. Antibodies used in Co-IP and the Western blots are marked in the figure. (G) Protein levels of Nucleolin, GFP-PE6, and GFP in 2% input used in Co-IP experiments.