Journal: Cell reports

Article Title: IPMK Mediates Activation of ULK Signaling and Transcriptional Regulation of Autophagy Linked to Liver Inflammation and Regeneration

doi: 10.1016/j.celrep.2019.02.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Beclin , Cell Signaling , 3738; RRID: AB_490837.

Techniques: Generated, Plasmid Preparation, Recombinant, Purification, SYBR Green Assay, DNA Purification, Staining, In Situ, shRNA, Software

Journal: Cancer Immunology Research

Article Title: Remodeling Translation Primes CD8+ T-cell Antitumor Immunity

doi: 10.1158/2326-6066.cir-19-0516

Figure Lengend Snippet: Figure 3. IL15-primed T cells increased translation in tumors. A, Flow cytometric analysis with quantification of protein synthesis or pS6 in IL2- or IL15-derived Pmel T cells (Thy1.1þ) transferred to tumor-bearing mice as described in Fig. 1D. Indicated organs were harvested 5 days later or harvested from transwell coculture with B16 tumor cells (B and C). D, Immunoblotting of T cells harvested as in B and C. E, Immunoblotting of IL2- or IL15-derived T cells treated with vehicle or increasing doses of U0126 p-p44/42 MAPK (Erk1/2) inhibitor. Flow cytometric analysis with quantification of protein synthesis (F) and pS6 in IL2- or IL15-derived T cells from transwell coculture with B16 tumors cells treated with vehicle or 10 mmol/L U0126 (G). Data are presented as SEM; , P < 0.05; , P < 0.01; , P < 0.001 (two-tailed t test). A, Data points represent individual mice. B–G, Data points represent biological replicates. Experiments in A–D performed three times and in E–G performed twice. CHX, cycloheximide; ns, not significant; Veh, vehicle.

Article Snippet: Protein concentrations were normalized using Pierce BCA Kit (Thermo Fisher Scientific, 23227) and loaded to 4%–10% agarose gels (Bio-Rad, 4568084). p-eEF2 (Thr56), eEF2, p-p70S6K (Thr389), p70S6K, p-AMPK (Thr172), AMPK, p-mTOR (S2448), p-p44/42 MAPK (Erk1/2) (Thr202/ Tyr204), Ubiquitin (P4D1), b-actin, and HRP-linked anti-rabbit and mouse secondaries were obtained from Cell Signaling Technology. p-p90RSK was obtained from BioLegend.

Techniques: Derivative Assay, Western Blot, Two Tailed Test

Journal: Hypertension

Article Title: Mitogen-Activated Protein Kinase–Activated Protein Kinase 2 in Angiotensin II–Induced Inflammation and Hypertension

doi: 10.1161/hypertensionaha.110.159889

Figure Lengend Snippet: Figure 3. A and B, Ang II increases p38 MAPK and MK2 phosphorylation levels. VSMCs were stimulated with vehicle (Veh) or Ang II for 10 minutes. Representative Western blots of phosphorylated and total p38 MAPK (A) and MK2 (B) and corresponding bar graphs are represented. C, VSMCs were transfected with a nontargeting control FITC-siRNA (siFITC) for 24 hours. Representative siFITC and 4,6- diamidino-2-phenylindole (DAPI) images are presented. D, MK2 siRNA transfection blunts Ang II–induced MK2 expression. VSMCs were transfected with siMK2 or negative control siRNA targeting luciferase (siLuc) and treated with Veh or Ang II for 24 hours. Repre- sentative Western blots of MK2 and bar graphs representing MK2 expression in different conditions are depicted. Results are meansSEM; n4 to 5; *P0.05 vs Veh for A and B and vs siLucVeh for D, and †P0.01 vs siLucAng II for D.

Article Snippet: Total or fractioned proteins (15 to 20 g) were extracted from VSMCs, separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated overnight at 4°C with antibodies (1:1000) against the following: MK2, p38 MAPK, and NF- B p65 subunit (from Cell Signaling Technology), and ICAM-1, VCAM-1, Ets-1, and p47phox (from Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Western Blot, Transfection, Control, Expressing, Negative Control, Luciferase