Journal: iScience

Article Title: The SH3-binding domain of chorismate mutase protein of Mycobacterium tuberculosis contributes to mycobacterial virulence

doi: 10.1016/j.isci.2024.111044

Figure Lengend Snippet: MtbCM interacts with AKAP9 and inhibits AKAP9-PKCε interaction in infected macrophages RAW 264.7 macrophages were infected with Msmeg-MtbCM at MOI of 1:50. After 5 h of infection cells were harvested, lysed and AKAP9 was immunoprecipitated (IP) using anti-AKAP9 Ab. The pull-down product was immunoblotted (IB) with anti-MtbCM Ab to detect MtbCM and total levels of AKAP9 and MtbCM in both the groups were shown as input controls (A). Results shown are representative of 3 different experiments. In another experiment, RAW 264.7 macrophages were infected with Msmeg-MtbCM at MOI of 1:50. After 5 h, cells were harvested, lysed and AKAP9 was immunoprecipitated (IP) using anti-AKAP9 Ab. The pull-down product was immunoblotted (IB) using anti-PKCε Ab to detect PKCε (B). Total levels of AKAP9, PKCε and MtbCM in both the groups were shown as input controls. Results shown are representative of 3 different experiments.

Article Snippet: Knock-down of AKAP9 in RAW 264.7 macrophages were performed using mouse specific AKAP9 siRNA (sc-45365) following the manufacturer’s protocol (Santa Cruz Biotechnology, USA).

Techniques: Infection, Immunoprecipitation

Journal: iScience

Article Title: The SH3-binding domain of chorismate mutase protein of Mycobacterium tuberculosis contributes to mycobacterial virulence

doi: 10.1016/j.isci.2024.111044

Figure Lengend Snippet: MtbCM localizes to Golgi network where it interacts with AKAP9 HEK-293T cells were transiently co-transfected with either pEGFP-MtbCM or pEGFP along with mCherry-TGOLN2. Cells were fixed using 4% formaldehyde and were observed under confocal microscope. DAPI was used to stain the nucleus (blue), (Scale bar: 1μm) (A). Colocalization coefficient was calculated using ZEN 2 (blue edition) software and was shown as Mean ± SEM from 50 cells of 3 independent experiments (B). In another experiment, RAW 264.7 macrophages were infected with either Msmeg-pVV or Msmeg-MtbCM at MOI of 1:50. Next, Golgi fractions were isolated. About 25 μg of PNS and Golgi fractions were resolved on SDS-PAGE and immunoblotted using either anti-AKAP9 Ab or anti-MtbCM Ab to show the presence of AKAP9 and MtbCM in the Golgi fraction (C). GAPDH was used as a negative control and GM130 was used as a Golgi marker. Next, AKAP9 was immunoprecipitated from cell lysates prepared from macrophages infected with Msmeg-pVV/Msmeg-MtbCM using anti-AKAP9 Ab and the pull-down product was immunoblotted using anti-MtbCM Ab to confirm AKAP9-MtbCM interaction (D). Results are representative of 3 independent experiments.

Article Snippet: Knock-down of AKAP9 in RAW 264.7 macrophages were performed using mouse specific AKAP9 siRNA (sc-45365) following the manufacturer’s protocol (Santa Cruz Biotechnology, USA).

Techniques: Transfection, Microscopy, Staining, Software, Infection, Isolation, SDS Page, Negative Control, Marker, Immunoprecipitation

Journal: iScience

Article Title: The SH3-binding domain of chorismate mutase protein of Mycobacterium tuberculosis contributes to mycobacterial virulence

doi: 10.1016/j.isci.2024.111044

Figure Lengend Snippet: SH3 binding domain of MtbCM is important for its interaction with AKAP9 and IL-10 signaling and survival of bacilli inside macrophages RAW 264.7 macrophages were infected with either Msmeg-MtbCM or Msmeg-MtbCM-PS4 with MOI of 1:50. After 5 h, cells were harvested, lysed and AKAP9 was immunoprecipitated (IP) with anti-AKAP9 Ab and the pull-down product was immunoblotted (IB) with either anti-MtbCM Ab or anti-PKCε Ab (A). The results are representative of 3 independent experiments. (B and C) Again, RAW 264.7 macrophages were left uninfected or infected with either Msmeg-pVV or Msmeg-MtbCM or Msmeg-MtbCM-PS4 at MOI 1:10. After 2 h of infection, cells were washed to remove extracellular bacilli and further incubated for another 3 h. Next cells were harvested and lysates were analyzed either for phosphorylated PKCε ( p -PKCε) and total PKCε (B) or for phosphorylated p38 MAPK (p-p38 MAPK) and total p38 MAPK (C) by Western blotting using specific combinations of primary and secondary antibodies. In another experiment, RAW 264.7 macrophages were infected with either Msmeg-pVV or Msmeg-MtbCM or Msmeg-MtbCM-PS4 at MOI of 1:10. After 24 h and 48 h, culture supernatants were collected for measuring IL-10 by two-site sandwich EIA (D). Results were shown as Mean ± SEM of 3 independent experiments. Also, RAW 264.7 macrophages were infected with either Msmeg-pVV or Msmeg-MtbCM or Msmeg-MtbCM-PS4 at MOI of 1:10. After 2 h of infection, cells were washed to remove extracellular bacilli and incubated for various time points, lysed and plated for CFU counts (E). In another experiment, RAW 264.7 macrophages were transfected with either control siRNA (AKAP-CT) or AKAP9 siRNA (AKAP-KD). After 24 h, cells were harvested, lysed and inhibition of AKAP9 protein levels was checked by Western blotting using anti-AKAP9 antibody and GAPDH was used as loading control (F). Next, AKAP-CT or AKAP-KD RAW 264.7 macrophages were infected with either Msmeg-pVV or Msmeg-MtbCM or Msmeg-MtbCM-PS4 with MOI of 1:10 or treated with medium alone (cells). After 2 h of infection, cells were washed to remove the extracellular bacilli and incubated further for 6 h. Cells were harvested and used for RNA isolation and cDNA synthesis and then qPCR was performed to find the mRNA levels of IL-10 (G). RPS29 transcript level was used as internal control. Results were shown as Mean ± SEM of 3 independent experiments. Unpaired t -test was used to calculate the p values.

Article Snippet: Knock-down of AKAP9 in RAW 264.7 macrophages were performed using mouse specific AKAP9 siRNA (sc-45365) following the manufacturer’s protocol (Santa Cruz Biotechnology, USA).

Techniques: Binding Assay, Infection, Immunoprecipitation, Incubation, Western Blot, Transfection, Control, Inhibition, Isolation, cDNA Synthesis

Journal: iScience

Article Title: The SH3-binding domain of chorismate mutase protein of Mycobacterium tuberculosis contributes to mycobacterial virulence

doi: 10.1016/j.isci.2024.111044

Figure Lengend Snippet:

Article Snippet: Knock-down of AKAP9 in RAW 264.7 macrophages were performed using mouse specific AKAP9 siRNA (sc-45365) following the manufacturer’s protocol (Santa Cruz Biotechnology, USA).

Techniques: Recombinant, Adjuvant, Protease Inhibitor, Transfection, Control, Cloning, Software, Microscopy

Journal: iScience

Article Title: The SH3-binding domain of chorismate mutase protein of Mycobacterium tuberculosis contributes to mycobacterial virulence

doi: 10.1016/j.isci.2024.111044

Figure Lengend Snippet: MtbCM interacts with AKAP9 and inhibits AKAP9-PKCε interaction in infected macrophages RAW 264.7 macrophages were infected with Msmeg-MtbCM at MOI of 1:50. After 5 h of infection cells were harvested, lysed and AKAP9 was immunoprecipitated (IP) using anti-AKAP9 Ab. The pull-down product was immunoblotted (IB) with anti-MtbCM Ab to detect MtbCM and total levels of AKAP9 and MtbCM in both the groups were shown as input controls (A). Results shown are representative of 3 different experiments. In another experiment, RAW 264.7 macrophages were infected with Msmeg-MtbCM at MOI of 1:50. After 5 h, cells were harvested, lysed and AKAP9 was immunoprecipitated (IP) using anti-AKAP9 Ab. The pull-down product was immunoblotted (IB) using anti-PKCε Ab to detect PKCε (B). Total levels of AKAP9, PKCε and MtbCM in both the groups were shown as input controls. Results shown are representative of 3 different experiments.

Article Snippet: AKAP9 siRNA (m) , Santa Cruz Biotechnology , Cat# sc-45365.

Techniques: Infection, Immunoprecipitation

Journal: iScience

Article Title: The SH3-binding domain of chorismate mutase protein of Mycobacterium tuberculosis contributes to mycobacterial virulence

doi: 10.1016/j.isci.2024.111044

Figure Lengend Snippet: MtbCM localizes to Golgi network where it interacts with AKAP9 HEK-293T cells were transiently co-transfected with either pEGFP-MtbCM or pEGFP along with mCherry-TGOLN2. Cells were fixed using 4% formaldehyde and were observed under confocal microscope. DAPI was used to stain the nucleus (blue), (Scale bar: 1μm) (A). Colocalization coefficient was calculated using ZEN 2 (blue edition) software and was shown as Mean ± SEM from 50 cells of 3 independent experiments (B). In another experiment, RAW 264.7 macrophages were infected with either Msmeg-pVV or Msmeg-MtbCM at MOI of 1:50. Next, Golgi fractions were isolated. About 25 μg of PNS and Golgi fractions were resolved on SDS-PAGE and immunoblotted using either anti-AKAP9 Ab or anti-MtbCM Ab to show the presence of AKAP9 and MtbCM in the Golgi fraction (C). GAPDH was used as a negative control and GM130 was used as a Golgi marker. Next, AKAP9 was immunoprecipitated from cell lysates prepared from macrophages infected with Msmeg-pVV/Msmeg-MtbCM using anti-AKAP9 Ab and the pull-down product was immunoblotted using anti-MtbCM Ab to confirm AKAP9-MtbCM interaction (D). Results are representative of 3 independent experiments.

Article Snippet: AKAP9 siRNA (m) , Santa Cruz Biotechnology , Cat# sc-45365.

Techniques: Transfection, Microscopy, Staining, Software, Infection, Isolation, SDS Page, Negative Control, Marker, Immunoprecipitation

Journal: iScience

Article Title: The SH3-binding domain of chorismate mutase protein of Mycobacterium tuberculosis contributes to mycobacterial virulence

doi: 10.1016/j.isci.2024.111044

Figure Lengend Snippet: SH3 binding domain of MtbCM is important for its interaction with AKAP9 and IL-10 signaling and survival of bacilli inside macrophages RAW 264.7 macrophages were infected with either Msmeg-MtbCM or Msmeg-MtbCM-PS4 with MOI of 1:50. After 5 h, cells were harvested, lysed and AKAP9 was immunoprecipitated (IP) with anti-AKAP9 Ab and the pull-down product was immunoblotted (IB) with either anti-MtbCM Ab or anti-PKCε Ab (A). The results are representative of 3 independent experiments. (B and C) Again, RAW 264.7 macrophages were left uninfected or infected with either Msmeg-pVV or Msmeg-MtbCM or Msmeg-MtbCM-PS4 at MOI 1:10. After 2 h of infection, cells were washed to remove extracellular bacilli and further incubated for another 3 h. Next cells were harvested and lysates were analyzed either for phosphorylated PKCε ( p -PKCε) and total PKCε (B) or for phosphorylated p38 MAPK (p-p38 MAPK) and total p38 MAPK (C) by Western blotting using specific combinations of primary and secondary antibodies. In another experiment, RAW 264.7 macrophages were infected with either Msmeg-pVV or Msmeg-MtbCM or Msmeg-MtbCM-PS4 at MOI of 1:10. After 24 h and 48 h, culture supernatants were collected for measuring IL-10 by two-site sandwich EIA (D). Results were shown as Mean ± SEM of 3 independent experiments. Also, RAW 264.7 macrophages were infected with either Msmeg-pVV or Msmeg-MtbCM or Msmeg-MtbCM-PS4 at MOI of 1:10. After 2 h of infection, cells were washed to remove extracellular bacilli and incubated for various time points, lysed and plated for CFU counts (E). In another experiment, RAW 264.7 macrophages were transfected with either control siRNA (AKAP-CT) or AKAP9 siRNA (AKAP-KD). After 24 h, cells were harvested, lysed and inhibition of AKAP9 protein levels was checked by Western blotting using anti-AKAP9 antibody and GAPDH was used as loading control (F). Next, AKAP-CT or AKAP-KD RAW 264.7 macrophages were infected with either Msmeg-pVV or Msmeg-MtbCM or Msmeg-MtbCM-PS4 with MOI of 1:10 or treated with medium alone (cells). After 2 h of infection, cells were washed to remove the extracellular bacilli and incubated further for 6 h. Cells were harvested and used for RNA isolation and cDNA synthesis and then qPCR was performed to find the mRNA levels of IL-10 (G). RPS29 transcript level was used as internal control. Results were shown as Mean ± SEM of 3 independent experiments. Unpaired t -test was used to calculate the p values.

Article Snippet: AKAP9 siRNA (m) , Santa Cruz Biotechnology , Cat# sc-45365.

Techniques: Binding Assay, Infection, Immunoprecipitation, Incubation, Western Blot, Transfection, Control, Inhibition, Isolation, cDNA Synthesis

Journal: iScience

Article Title: The SH3-binding domain of chorismate mutase protein of Mycobacterium tuberculosis contributes to mycobacterial virulence

doi: 10.1016/j.isci.2024.111044

Figure Lengend Snippet:

Article Snippet: AKAP9 siRNA (m) , Santa Cruz Biotechnology , Cat# sc-45365.

Techniques: Recombinant, Adjuvant, Protease Inhibitor, Transfection, Control, Cloning, Software, Microscopy