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 affects the PKCε-IRAK3-MKP-1 signaling cascades in macrophages RAW 264.7 macrophages were either treated with medium alone (cells) or infected with either Msmeg-pVV or Msmeg-MtbCM at MOI of 1:10. After 2 h of infection cells were washed to remove extracellular bacilli and incubated further for 3 h. Cells were lysed and the levels of MKP-1 were examined by Western blotting using anti-MKP-1 Ab (A). GAPDH was used as loading control and densitometric quantification of Western blots was performed using ImageJ software (B). Results shown are Mean ± SEM of 3 independent experiments. In another experiment, RAW 264.7 macrophages were infected with either Msmeg-pVV or Msmeg-MtbCM at 1:10 MOI. After 2 h of infection, cells were washed to remove extracellular bacilli and further incubated for another 3 h. Next cells were washed, fixed with 4% paraformaldehyde followed by permeabilization using 0.1% Triton X-100 and stained with anti-IRAK3 Ab at a dilution of 1:1000. Next, cells were washed and incubated with Alexa Flour-488 conjugated goat anti-rabbit secondary Ab at a dilution of 1:1000. After mounting with DAPI containing mounting medium, cells were observed under confocal microscope (C), (Scale bar: 5 μm). Results shown are representative of 3 different experiments. Also, lysates in Figure 3 A were used to check the levels of phospho-PKCε and total PKCε by Western blotting using anti-phospho-PKCε Ab and total PKCε Ab respectively (D) and densitometric quantification of Western blot was performed using ImageJ software (E). Results shown are Mean ± SEM of 3 independent experiments. Unpaired t -test was used to calculate the p values for Figures 3 B and 3E. In another experiment, RAW 264.7 macrophages were either treated with medium alone (cells) or infected with either Msmeg-pVV or Msmeg-MtbCM with MOI of 1:10 in the absence or presence of DCP-LA (100 nM). After 2 h of infection, cells were washed with 1X PBS to remove extracellular bacilli and incubated for another 2 h. Cells were harvested and protein lysates were prepared and analyzed for phosphorylated p38 MAPK (p-p38 MAPK) and total p38 MAPK (F) as well as MKP-1 (G) protein levels by Western blotting using specific antibodies. GAPDH was used as a loading control. Results shown are representative of 3 different experiments.

Article Snippet: Rabbit anti-IRAK3 Ab , Cell Signaling Technology, USA , Cat# 4369.

Techniques: Infection, Incubation, Western Blot, Control, Software, Staining, 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:

Article Snippet: Rabbit anti-IRAK3 Ab , Cell Signaling Technology, USA , Cat# 4369.

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

Journal: EMBO Molecular Medicine

Article Title: Immune-enhancing neutrophils reprogrammed by subclinical low-dose endotoxin in cancer treatment

doi: 10.1038/s44321-024-00100-7

Figure Lengend Snippet: Purified neutrophils were treated with 100 pg/ml LPS or PBS as control overnight. Protein lysate were subjected to immunoblotting analysis. ( A ) Protein levels of pSTAT5 and STAT5, and relative expressions were normalized to β-actin. n = 4. ( B ) Protein levels and relative expressions of PSMD10. n = 4. ( C ) Protein levels and relative expressions of IRAK-M. n = 3. ( D ) Protein levels and relative expressions of KLF4. n = 4. ( E , F ) Co-immunoprecipitation analysis of IRAK-M with STAT3 and STAT1 from neutrophil lysates. ( G , H ) Protein levels of STAT3 and STAT1 in cytoplasm and nucleus. Quantification of nucleus STAT1/3 expressions normalized to histone H3. n = 3. Data information: Data ( A – D , and G , H ) are represented as means ± SD of independent biological replicates ( n ). Significance was calculated by unpaired two-tailed student t test. .

Article Snippet: The whole-cell lysates were utilized for immunoprecipitation with the anti-IRAK-M antibody following the manufacturer’s instructions, supported by Pierce™ Classic Magnetic IP/Co-IP Kit (ThermoFisher, catalog no. 88804).

Techniques: Purification, Control, Western Blot, Immunoprecipitation, Two Tailed Test