phospho 4ebp1 thr37 46 rabbit mab  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Arginine regulates the mucoid phenotype of hypervirulent Klebsiella pneumoniae

doi: 10.1038/s41467-025-61047-y

Figure Lengend Snippet: A Macrophages (BEI NR-9456) were incubated with KPPR1 or ∆ argR for 2 hr (MOI = 10). Internalization was determined by a 1 hr gentamicin treatment. Macrophages were washed with PBS, lysed with 0.2% TritonX100, and output CFUs were enumerated and normalized to input. Experiments were performed > 3 independent times, in triplicate ( n = 9). Data presented are the mean, and error bars represent the standard error of the mean. B C57BL/6 mice were infected retropharyngeally with 1 × 10 6 CFUs of a 1:1 mixture of WT + EV and either ∆ argR + EV or ∆ argR + p argR . The log 10 competitive index at 24 h post-infection is shown relative to 0. Each dot represents an individual mouse (open symbols = male; half-filled = female). The horizontal line represents the median; box bounds indicate the minima (25 TH percentile) and maxima (75 TH percentile); and whiskers represent the full range. ∆ argR + EV was tested in two independent experiments ( n = 8), while ∆ argR +p argR was tested once ( n = 7). C Arginine concentrations were measured in murine lungs, spleens, livers, and feces. Samples were collected from three mice and experiments were performed 3 independent times, in technical triplicate ( n = 3). Data presented are the mean, and error bars represent the standard error of the mean. Statistical significance was determined using an unpaired two-tailed t -test ( A ), a two-tailed unpaired t -test ( B , comparing CI values between ∆ argR + EV and ∆ argR +p argR ), and a two-tailed one-sample t -test ( B , comparing CI to a hypothetical value of zero). In ( C ), a one-way ANOVA with Tukey’s post-test was used. p -values are displayed above each comparison; * p < 0.05; ** p < 0.01, *** p < 0.001; # p < 0.0001. Exact P- values: ( A ) left to right: <0.0001, 0.0287; (B) left to right above data bars: 0.0008, 0.0078, 0.0081; connected by lines: <0.0001; ( C ) left to right: 0.0033, <0.0001, 0.0327, 0.0003. Source data are provided as a Source Data file.

Article Snippet: Immortalized macrophage cells derived from WT mice (BEI Resources #NR-9456) were maintained in DMEM medium with L-glutamine, 4.5 g/L glucose and sodium pyruvate (Corning) supplemented with 10% heat-inactivated fetal calf serum (Corning), 100 U/mL penicillin, and 100 μg/mL streptomycin in an atmosphere of 5% CO 2 .

Techniques: Incubation, Infection, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Arginine regulates the mucoid phenotype of hypervirulent Klebsiella pneumoniae

doi: 10.1038/s41467-025-61047-y

Figure Lengend Snippet: When cultured in the presence of arginine, K. pneumoniae increases mucoidy without altering the total capsule abundance. When arginine is brought into the cell, the arginine regulator, ArgR, binds arginine and becomes active. The activated ArgR-arginine complex acts as a transcriptional regulator via binding to ARG boxes. The arginine-ArgR complex binds directly to the P rmp ARG box and activates the rmpADC promoter, upregulating rmpADC transcription. The RmpD protein then interacts with the Wzc protein, a tyrosine autokinase that regulates high-level capsular polysaccharide (CPS) polymerization. RmpD-Wzc interactions decrease ‘Type A’ polysaccharide chains and increases ‘Type B’, which decreases CPS diversity and presents as the mucoid phenotype resulting in decreased association with macrophages. Created in BioRender. Ryan, B. (2025) https://BioRender.com/r74w727 .

Article Snippet: Immortalized macrophage cells derived from WT mice (BEI Resources #NR-9456) were maintained in DMEM medium with L-glutamine, 4.5 g/L glucose and sodium pyruvate (Corning) supplemented with 10% heat-inactivated fetal calf serum (Corning), 100 U/mL penicillin, and 100 μg/mL streptomycin in an atmosphere of 5% CO 2 .

Techniques: Cell Culture, Binding Assay

Journal: Journal of Innate Immunity

Article Title: TLR2 or TLR4 Stimulation Induces Transmembrane TNF-Driven Priming in Macrophages Which Results in Improved Clearance of a Subsequent Staphylococcus aureus Infection

doi: 10.1159/000546011

Figure Lengend Snippet: TLR-induced TNF is required for improved macrophage clearance of S. aureus. a WT BMDMs, ( b ) TLR2−/− and TLR4−/− BMDMs were treated with either 1 μg/mL PAM2, LPS, or equal volume PBS for 12 h followed by S. aureus infection for 5 h (MOI 2:1). Bacterial burdens (CFUs) were determined after overnight incubation on TSA plates. Results shown in panels A and B were obtained from 5 independent experiments per cell line. Each data point represents the average of two technical replicates per experiment. A ROUT outlier test was performed and identified outliers were removed from the datasets prior to statistical analysis. Statistical analysis was performed with ordinary one-way ANOVA: *** p = 0.0009 for PBS vs. PAM2-treated WT BMDMs, *** p = 0.0003 for PBS vs. LPS treated WT BMDMs), * p = 0.0148 for PBS vs. PAM2-treated TLR4−/− BMDMs). c WT mice were intratracheally inoculated with 100 μL of either 10 μg PAM2, PAM3, or equal volume PBS. Lung homogenates collected 6 h post-inoculation were processed and analyzed by RNA sequencing. Volcano plots were generated in R studio. d–f TNF transcript 3 h following PAM2 or LPS stimulation of immortalized murine WT BMDMs ( d ), TLR2−/− BMDMs ( e ), and TLR4−/− BMDMs ( f ). Data were normalized to reference gene β-actin, and fold change was calculated using ΔΔCT method. Results were obtained from 3 independent experiments that had three technical replicates each. Each data point represents the average of three technical replicates. g Primary TNF−/− BMDMs were treated with either 1 μg/mL PAM2, LPS, or equal volume PBS for 12 h followed by S. aureus infection for 5 h (MOI 2:1). Bacterial burdens (CFUs) were determined after overnight incubation on TSA plates. Data were obtained from BMDMs isolated from 5 mice. BMDMs from each mouse were run as an independent assay which contained 2 technical replicates. Each data point represents the average of two technical replicates.

Article Snippet: Murine immortalized macrophages (cell line NR-9456, BEI Resources; Manassas, VA, USA) were grown and maintained as previously described until 90% confluency was achieved.

Techniques: Infection, Incubation, RNA Sequencing, Generated, Isolation

Journal: bioRxiv

Article Title: MAPK and mTORC1 signaling converge on Cyclin D to enable cell-cycle re-entry in melanoma persister cells

doi: 10.1101/2025.05.14.653869

Figure Lengend Snippet: (A) MSigDB hallmark gene set enrichment analysis of 40 differentially expressed genes from scRNA-sequencing dataset comparing 1 µM dabrafenib-treated escapees to non-escapees and untreated cycling cells. A false-discovery rate cutoff of 0.05 was used. Data replotted from (B) Signaling diagram of mTORC1 pathway on growth and translation vs. autophagy. (C) Representative IF images of either A375, SKMEL28, or WM278 cells treated with 1 µM dabrafenib for 72 h and co-stained for Hoescht, p-Rb (S780), and various mTORC1 activity markers (p-eiF4e (S209), p-S6 (S240/244), p-4EBP1 (Thr37/46)), and translation rate (using OPP). Quantification of each marker is plotted as bulk violin plots in untreated cells (gray) vs dabrafenib-treated (purple) cells, as well as split violin plots in dabrafenib-treated non-escapees (p-Rb - , blue) vs. escapees (p-Rb + , pink). Scalebar = 50 μm. White arrows highlight representative escapee cells. (D) Split violin plots quantifying two mTORC1 activity markers in non-cycling (p-Rb - , blue) vs cycling A375 cells (p-Rb + , pink) treated for 72 h with varying drug conditions. UT, untreated. (E) Representative IF images and split violin plots of untreated and 1 µM dabrafenib-treated A375 cells quantifying lysosomal marker LAMP1 in non-escapees (p-Rb - , blue) vs escapees (p-Rb + , pink). Scalebar = 50 μm. (F) Autophagic flux measured in UT and 1 µM dabrafenib-treated A375 by change in LC3II after a 3 h treatment with 10 μM chloroquine. Error bars: mean ± std of four replicate wells. For bar graphs, p -values were calculated by unpaired t-tests between indicated drug conditions, specified with a black line linking the conditions. For bulk violins, p -values were determined by Mann-Whitney U-tests between indicated drug conditions, specified with a black line linking the conditions. For split violins, p -values were determined by Mann-Whitney U-tests between non-escapee (p-Rb - ) compared to escapee cells (p-Rb + ).

Article Snippet: Primary antibodies and dilutions used for this study include: phospho-Rb (Ser807/811) (D20B12) rabbit mAb (1:500 IF, CST #8516); phospho-Rb (Ser780) mouse mAb (1:1000 IF, BD Biosciences #558385); phospho-ERK1/2 (Thr202/Tyr204) rabbit mAb (1:500 IF, CST #4370); phospho-ERK1/2 (Thr202/Tyr204) mouse mAb (1:1000 IF, MilliporeSigma #M9692); phospho-S6 (Ser240/244) rabbit mAb (1:500 IF, CST #2215); phospho-eIF4e (Ser209) (EP2151Y) rabbit mAb (1:400 IF, Abcam, #ab76256); phospho-4EBP1 (Thr37/46) rabbit mAb (1:250 IF CST # 9456S); LAMP1 D2D11 XP rabbit mAb (1:1000 IF, CST #9091); LC3 II rabbit mAb (1:1000 IF, Abcam, #ab192890); Anti-Cyclin D1 clone SP4 rabbit mAb (1:500 IF Thermo Fisher, RM-9140-S0); Histone H3 mouse mAb (1:2000 WB, CST, #3638); phospho-AKT (Ser473) rabbit mAb (1:200 IF, CST, #9271).

Techniques: Sequencing, Staining, Activity Assay, Marker, MANN-WHITNEY

Journal: eLife

Article Title: Ezrin defines TSC1 activation at endosomal compartments through EGFR-AKT signaling

doi: 10.7554/elife.98523

Figure Lengend Snippet: Figure 6. EGFR–Ezrin complex interacts with TSC1. (a) Co-IP analysis for Ezr–TSC1 (left) and EGFR–TSC1 (right) interaction. For co-IP analyses, Ezrin (left) and EGFR (right) antibodies were used. The proteins immunoprecipitated were blotted for TSC1 and AKT antibodies in HeLa WT and EZR−/−. (b) HeLa WT and EZR−/− cells were lysed and immunoblotted with pS939 TSC2, TSC2, PT389 P70 S6 Kinase, P70 S6 Kinase, pS473 AKT, AKT, pS65 4E-BP1, 4E-BP1, and GAPDH as a loading control. Data represent the mean of pS939 TSC2/TSC2, T389 P70 S6 Kinase/P70 S6 Kinase, pS473 AKT/AKT, and pS65 4E-BP1/4E-BP1 ratio ± SEM (n = 3 experiments at least). Statistical test: unpaired t-test for pT389 P70 S6 Kinase, pS473 AKT; unpaired t-test with Welch’s correction for pS939 TSC2; Mann–Whitney test for pS939 TSC2. (c) pP70 S6 Kinase western blotting with insulin time course in HeLa WT

Article Snippet: For western blot analysis, the following antibodies were used: mouse anti- NBR1 (1:1000, Abnova MO1), rabbit anti- LAMP1 (1:500, Sigma L1418), mouse anti- Ezrin (1:1000, Novex 357300), mouse anti- SQSTM1/P62 (1:1000, Abcam ab56416), rabbit anti- Cathepsin D (1:1000, Cell Signaling 2284), rabbit anti- LC3 (1:1000, Novus NB100- 2220), mouse anti- GAPDH (1:1000, Santa Cruz SC- 32233), rabbit anti- HER2/ErbB2 (1:1000, Cell Signaling 2165), rabbit anti- HER3/ErbB3 (1:1000, Cell Signaling 12708), rabbit anti- phospho- EGF receptor (Tyr845) (1:1000, Cell Signaling 6963), rabbit anti- EGF receptor (1:1000, Cell Signaling 4267), rabbit anti- MAPKAPK- 2 (1:1000, Cell Signaling 3042), rabbit anti- phospho- MAPKAPK- 2 (Thr222) (1:1000, Cell Signaling 3316), rabbit antip38 MAPK (1:1000, Cell Signaling 8690), rabbit anti- phospho- p38 MAPK (Thr180/Tyr182) (1:1000, Cell Signaling 4511), rabbit anti- ZO1 (1:1000, Abcam ab216880), mouse anti- EEA1 (1:1000, BD 610457), rabbit anti- Tuberin/TSC2 (1:1000, Cell Signaling 4308), rabbit anti- phospho- Tuberin/TSC2 (Ser939) (1:1000, Cell Signaling 3615), rabbit anti- phospho- Tuberin/TSC2 (Thr1462) (1:1000, Cell Signaling 3617), rabbit anti- p70 S6 Kinase (1:1000, Cell Signaling 9202), mouse anti- phospho- p70 S6 Kinase (Thr389) (1:1000, Cell Signaling 9206), rabbit anti- Akt (1:1000, Cell Signaling 9272), rabbit anti- phospho- Akt (Ser473) (1:1000, Cell Signaling 4060), rabbit anti- 4E- BP1 (1:1000, Cell Signaling 9644), rabbit anti- phospho- 4E- BP1 (Ser65) (1:1000, Cell Signaling 9456), rabbit anti- phospho- 4E- BP1 (Thr37/46) (1:1000, Cell Signaling 2855), rabbit anti- Hamartin/TSC1 (1:1000, Cell Signaling 6935), mouse anti- EGFR (1:500, Santa Cruz sc- 120), mouse anti- p- EGFR (1:500, Santa Cruz sc- 57542).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Control, MANN-WHITNEY, Western Blot

Journal: eLife

Article Title: Ezrin defines TSC1 activation at endosomal compartments through EGFR-AKT signaling

doi: 10.7554/elife.98523

Figure Lengend Snippet: Figure 7. Ezrin depletion induces EGFR-mediated retinal degeneration. (a) Schematic representation of used CRISPR/Cas9 strategy to generate Ezrin−/− medaka lines. The red box highlighted the deleted nucleotides in the Ezrin exon 1 gene. (b) WT and Ezrin−/− medaka proteins were immunoblotted with Ezrin antibody and Actin as a loading control. (c) Stereo-microscopic representative images of WT and Ezrin−/− medaka at stage 40. Scale bar: 1 mm. (d) Immunoblots and calculated levels (right) of pT1462 TSC2, pS473 Akt, LC3-I, LC3-II, pY845 Egfr and Egfr, pS65 4E-BP1 in WT and Ezrin−/− medaka

Article Snippet: For western blot analysis, the following antibodies were used: mouse anti- NBR1 (1:1000, Abnova MO1), rabbit anti- LAMP1 (1:500, Sigma L1418), mouse anti- Ezrin (1:1000, Novex 357300), mouse anti- SQSTM1/P62 (1:1000, Abcam ab56416), rabbit anti- Cathepsin D (1:1000, Cell Signaling 2284), rabbit anti- LC3 (1:1000, Novus NB100- 2220), mouse anti- GAPDH (1:1000, Santa Cruz SC- 32233), rabbit anti- HER2/ErbB2 (1:1000, Cell Signaling 2165), rabbit anti- HER3/ErbB3 (1:1000, Cell Signaling 12708), rabbit anti- phospho- EGF receptor (Tyr845) (1:1000, Cell Signaling 6963), rabbit anti- EGF receptor (1:1000, Cell Signaling 4267), rabbit anti- MAPKAPK- 2 (1:1000, Cell Signaling 3042), rabbit anti- phospho- MAPKAPK- 2 (Thr222) (1:1000, Cell Signaling 3316), rabbit antip38 MAPK (1:1000, Cell Signaling 8690), rabbit anti- phospho- p38 MAPK (Thr180/Tyr182) (1:1000, Cell Signaling 4511), rabbit anti- ZO1 (1:1000, Abcam ab216880), mouse anti- EEA1 (1:1000, BD 610457), rabbit anti- Tuberin/TSC2 (1:1000, Cell Signaling 4308), rabbit anti- phospho- Tuberin/TSC2 (Ser939) (1:1000, Cell Signaling 3615), rabbit anti- phospho- Tuberin/TSC2 (Thr1462) (1:1000, Cell Signaling 3617), rabbit anti- p70 S6 Kinase (1:1000, Cell Signaling 9202), mouse anti- phospho- p70 S6 Kinase (Thr389) (1:1000, Cell Signaling 9206), rabbit anti- Akt (1:1000, Cell Signaling 9272), rabbit anti- phospho- Akt (Ser473) (1:1000, Cell Signaling 4060), rabbit anti- 4E- BP1 (1:1000, Cell Signaling 9644), rabbit anti- phospho- 4E- BP1 (Ser65) (1:1000, Cell Signaling 9456), rabbit anti- phospho- 4E- BP1 (Thr37/46) (1:1000, Cell Signaling 2855), rabbit anti- Hamartin/TSC1 (1:1000, Cell Signaling 6935), mouse anti- EGFR (1:500, Santa Cruz sc- 120), mouse anti- p- EGFR (1:500, Santa Cruz sc- 57542).

Techniques: CRISPR, Control, Western Blot

Journal: eLife

Article Title: Ezrin defines TSC complex activation at endosomal compartments through EGFR–AKT signaling

doi: 10.7554/eLife.98523

Figure Lengend Snippet:

Article Snippet: Antibody , anti-phospho-4E-BP1 (Ser65) (rabbit monoclonal) , Cell Signaling , 9456 , WB (1:1000).

Techniques: CRISPR, Transfection, Construct, Sequencing, Recombinant, Purification, Western Blot, Software, Mass Spectrometry, Immunofluorescence, Functional Assay, Fluorescence, Imaging, Staining, Plasmid Preparation