Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for IL-15-driven B-CLL cycling: Roles for AKT and STAT5 in modulating Cyclin D2 and DNA damage response proteins 1

doi: 10.4049/jimmunol.1801142

Figure Lengend Snippet: B-CLL cells were pre-cultured for 20 h in medium ± ODN prior to adding IL-15. After varying intervals, pAKTSer473 levels were monitored by immunofluorescent staining and flow cytometry. (A) Fluorescence histograms of cytoplasmic pAKT in viable-gated U-CLL996 cells at 30 min post-IL-15 (left column) or cytoplasmic + nuclear levels of pAKT in U-CLL1158 cells at 60 min post-IL-15 (right column) (see Materials & Methods for procedural differences). Inserted values represent RMFI (ratio of median fluorescence intensity (MFI) in anti-pAKT-stained versus isotype control-stained cells). (B) Fold-increase in pAKT fluorescence (ratio: +IL15/no IL-15) within medium-pre-cultured cells (left) and ODN-primed cells (right) at varying intervals (15 to 1200 min) post IL-15. Center legend shows individual B-CLL tested (U-CLL = open symbols; M-CLL = closed symbols). (C) Bar graphs summarizing fold-increase in pAKT levels in ODN-primed or unprimed B-CLL at varying intervals after IL-15. Statistical significance determined by 2-sided, paired t-test. Note: Values for baseline pAKT fluorescence in medium- and 20h ODN-primed B-CLL with no IL-15 exposure were 259±131 and 266±126 (mean ± SD); not significantly different by statistical analysis.

Article Snippet: Levels of activated AKT and the above STAT family members were measured by intracellular staining with specific fluorochrome-conjugated mAbs: (a) AF-647-rabbit anti-pAKT Ser-473 mAb (cat #2337; Cell Signaling Technology), with rabbit IgG control (each at 0.05 μg/ml), or alternatively AF-647 or AF-488-conjugated mouse anti-pAKT Ser-473 (BD-Biosciences), with mouse IgG1 control (Santa-Cruz) (each at 1.5 μg/ml). (b) AF-647-mouse anti-STAT5 Y694/699 (mAb clone 47; BD-Biosciences), with mouse IgG1 control (Santa Cruz) (3 μg/ml).

Techniques: Cell Culture, Staining, Flow Cytometry, Fluorescence

Journal: Scientific Reports

Article Title: Stability of tuberous sclerosis complex 2 is controlled by methylation at R1457 and R1459

doi: 10.1038/s41598-020-78274-6

Figure Lengend Snippet: Arginine methylation of TSC2 blockes Akt-dependent TSC2 phosphorylation at T1462. ( A ) Phosphorylation assays were conducted using the unmodified TSC2 peptide (cont) and the modified arginine methylated peptide (Methyl). Peptides were incubated with recombinant Akt and [γ- 32 P] ATP and phosphorylation was detected using a scintillation counter. Error bars indicate standard deviation (SD) from three independent experiments; ** p < 0.01. ( B/C ) Effect of TSC2 methylation on TSC2 phosphorylation measured by inhibiting ( B ) [ 3 H]-SAM using cycloleucine (100 mM) with MG132 (10 µM) or ( C ) PRMT1 activity using AMI-5 (2.0 µM). Phosphorylated TSC2 (T1462) and AKT (S473) levels were analysed by western blotting. Error bars indicate the SD from two independent experiments; * p < 0.05. All experiments were repeated three times.

Article Snippet: The following primary antibodies were used and diluted in 5% BSA/TBST: anti-PRMT1 polyclonal antibody (Cell Signaling Technology (CST), Cat. #2449), anti-TSC2 monoclonal antibody (CST, Cat. #4308), anti-phospho-TSC2 (T1462) monoclonal antibody (CST, Cat. #3611), anti-β-actin monoclonal antibody (Santa Cruz, Cat. #sc-47778), anti-Akt polyclonal antibody (CST, Cat. #9272), anti-phospho-Akt (S473) monoclonal antibody (CST, Cat. #4051), anti-asymmetric di-methyl arginine motif (adme-R) MultiMab Rabbit mAb mix antibody (CST, Cat. #13522), anti-HA HA-7 monoclonal antibody (Sigma, Cat. #H2663), anti-FLAG M2 monoclonal antibody (Sigma, Cat. #F3165), and anti-Myc My3 monoclonal antibody (MBL International, Cat. #M192-3).

Techniques: Methylation, Modification, Incubation, Recombinant, Standard Deviation, Activity Assay, Western Blot