Journal: Oncotarget

Article Title: Initiation of tumor dormancy by the lymphovascular embolus

doi: 10.18632/oncotarget.28658

Figure Lengend Snippet: Westen blots of mTOR activity with spontaneous spheroidgenesis of Mary-X as measured by pmTOR (Ser2481) and pmTOR (Ser2448) ( A ); of downstream mTOR substrate p-p70-S6K(Thr389) activity in Mary-X spheroids ( B ); of mTOR activity as measured by pmTOR(Ser2448) in induced MCF-7 spheroids ( C ); of downstream mTOR substrates p-p70-S6K(Thr389), p-p70-S6K(Ser371) and p4E-BP1 in induced MCF-7 spheroids; ( D ); of pmTOR(Ser2448) and downstream mTOR substrates p-p70-S6K(Thr389) and p4E-BP1in induced MCF-7 spheroids ( E ).

Article Snippet: We used the following antibodies for Western blot studies: PI3K Ab Sampler Kit (1:1000 dilution, Rabbit, #9655, Cell Signaling Technology (CST)), AMPK and ACC Antibody Sampler Kit (1:1000 dilution, Rabbit, #9957, (CST)), mTOR Substrates Antibody Sampler Kit (1:1000 dilution, Rabbit, #9862, (CST)), E-Cadherin (24E10) (1:1000 dilution, Rabbit mAb #3195, (CST)) and Calpain 2 Large Subunit (M-type) (1:1000 dilution, #3195, (CST)).

Techniques: Activity Assay

Journal: Oncotarget

Article Title: Initiation of tumor dormancy by the lymphovascular embolus

doi: 10.18632/oncotarget.28658

Figure Lengend Snippet: Westen blots of mTOR and AMPK activities and their correlation with levels of calpain 2 and E-cad/NTF1 in spontaneous spheroidgenesis of Mary-X ( A ); of calpain 2 and E-cad/NTF1 and their correlation with the stages of induced spheroidgenesis of E-cadherin positive MCF-7 cells ( B ); of calpain 2 and its correlation with the stages of induced spheroidgenesis of E-cadherin negative MDA-MB-468 cells ( C ). Westen blots of the effects of calpain inhibition on mTOR and AMPK activities in MCF-7 cells grown as monolayers ( D ) and as induced spheroids ( E ).

Article Snippet: We used the following antibodies for Western blot studies: PI3K Ab Sampler Kit (1:1000 dilution, Rabbit, #9655, Cell Signaling Technology (CST)), AMPK and ACC Antibody Sampler Kit (1:1000 dilution, Rabbit, #9957, (CST)), mTOR Substrates Antibody Sampler Kit (1:1000 dilution, Rabbit, #9862, (CST)), E-Cadherin (24E10) (1:1000 dilution, Rabbit mAb #3195, (CST)) and Calpain 2 Large Subunit (M-type) (1:1000 dilution, #3195, (CST)).

Techniques: Inhibition

Journal: Oncotarget

Article Title: Initiation of tumor dormancy by the lymphovascular embolus

doi: 10.18632/oncotarget.28658

Figure Lengend Snippet: Westen blots of the effects of different pathway inhibitors including rapamycin (mTOR), LY294002 (P13K) and U0126 (MAPK) on mTOR activity in Mary-X spontaneous spheroidgenesis ( A ); of the opposite effects of LY294002 (P13K) on mTOR and AMPK activities in both MCF-7 monolayers (left) and induced spheroids (right) ( B ); of P13K activity during induced spheroidgenesis of MCF-7 cells ( C ); of the differential effects of calpain inhibition on P13K activity in MCF-7 monolayers (left) v induced spheroids (right) ( D ). β-Tubulin housekeeping probe was used to normalize for protein loading on divided blots depicted in , as well as . ACTB housekeeping probe was used to normalize for protein loading on divided blots depicted in , as well as , left and right.

Article Snippet: We used the following antibodies for Western blot studies: PI3K Ab Sampler Kit (1:1000 dilution, Rabbit, #9655, Cell Signaling Technology (CST)), AMPK and ACC Antibody Sampler Kit (1:1000 dilution, Rabbit, #9957, (CST)), mTOR Substrates Antibody Sampler Kit (1:1000 dilution, Rabbit, #9862, (CST)), E-Cadherin (24E10) (1:1000 dilution, Rabbit mAb #3195, (CST)) and Calpain 2 Large Subunit (M-type) (1:1000 dilution, #3195, (CST)).

Techniques: Activity Assay, Inhibition

Journal: Oncotarget

Article Title: Initiation of tumor dormancy by the lymphovascular embolus

doi: 10.18632/oncotarget.28658

Figure Lengend Snippet: TMAs ( A ) were subjected to ERAs and SRAs designed to measure true lymphovascular tumor emboli and distinguish them from tumor clumps showing separation artefact from adjacent stroma ( A – C ). Imaging strategy was predicated on SRA’s recognizing podoplanin (D2-40) and CD31 red colorimetric immunoreactivities (B) and the use of ERA’s recognizing epithelial clustering algorithms eg, the Gaussian kernel ( D ), which selectively recognized the clusters within lymphovascular spaces and imaged them ( E ). SRAs showed, compared to their respective non-embolic areas ( F – I ), increased E-cadherin ( J ), decreased Ki-67 ( K ), decreased mTOR (Serine2481) ( L ) and decreased mTOR (Serine2448) ( M ) signal intensities within the emboli of both IBC and non-IBC cases ( N ). For each of these parameters, the graph depicts calculated means ± SD ( N ). Differences of significance are depicted.

Article Snippet: We used the following antibodies for Western blot studies: PI3K Ab Sampler Kit (1:1000 dilution, Rabbit, #9655, Cell Signaling Technology (CST)), AMPK and ACC Antibody Sampler Kit (1:1000 dilution, Rabbit, #9957, (CST)), mTOR Substrates Antibody Sampler Kit (1:1000 dilution, Rabbit, #9862, (CST)), E-Cadherin (24E10) (1:1000 dilution, Rabbit mAb #3195, (CST)) and Calpain 2 Large Subunit (M-type) (1:1000 dilution, #3195, (CST)).

Techniques: Imaging

Journal: NPJ precision oncology

Article Title: Taurine and proline promote lung tumour growth by co-regulating Azgp1/mTOR signalling pathway.

doi: 10.1038/s41698-025-00872-2

Figure Lengend Snippet: Fig. 7 | Azgp1/mTOR axis is identified as an under-reported pathway for taurine and proline in promoting lung cancer progression. Taurine and proline may inhibit AZGP1 function, triggering mTOR pathway activation and lipid metabolism disorder that ultimately promote lung cancer progression.

Article Snippet: The following antibodies were used in this experiment: β-Actin (Cell Signalling Technology (CST), #8457), p-mTOR (Santa Cruz, sc-293133), mTOR (CST, #2983), Azgp1 (ZAP) (Santa Cruz, sc-21720).

Techniques: Activation Assay

Journal: Immunity

Article Title: Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8 + T Cells.

doi: 10.1016/j.immuni.2018.02.012

Figure Lengend Snippet: Figure 3. mTORC2, AKT, and GSK3b Signaling Components Are Present in Mitochondria-ER Junctions (A) Immunoblots of MEJ, PM, and EGC fractions and total lysate (T) from Jurkat T cells. Blots were probed with mTOR, RICTOR, RAPTOR, and AKT antibodies. COX IV was used as loading and fraction-validation control (representative of n = 3 independent experiments). (B) Immunoblots of EGC and MEJ fractions from NV and EM human CD8+ T cells probed with antibodies specific for RICTOR and mTOR. GRP75 was used to validate the respective fractions (representative of n = 2 independent samples, each sample consisted of 4 3 107 sorted cells, pooled from 2–4 donors). (C) Immunoblot analysis of EGC and MEJ fractions from bulk CD8+ T cells treated with non-loaded control beads (–) or activated with a-CD3 and a-CD28 mAb-loaded beads (+) for 2 hr. Blots were probed with mTOR and RICTOR antibodies. Cox IV was used as fraction validation control (representative of n = 3 independent experiments). (D) Left, immunoblot analysis of MEJ fractions from wild-type (WT) and rictor KO memory CD8+ T cells re-stimulated with OVA peptide for 1 hr (+) or from non-activated counterparts (–). Blots were probed for total Akt, pAkt-S473, pAkt-T308, and Cox IV. Right, quantification was performed by normalization of targets to Cox IV. Bar graphs show fold change in Akt phosphorylation following activation relative to non-activated controls (n = 3 independent experiments). (E and F) Summary of metabolic flux analysis on EM CD8+ T cells activated and assayed as in Figure 1A. Cells were treated with inhibitors of mTOR (OSI-027, 10 mM, and KU0063794, 10 mM) (E) or AKT (Akti, 10 mM, and MK2206, 10 mM) (F). Bar graphs show basal and maximal respiration (n = 4–8 donors). (G) Immunoblot analysis of total cell lysates from EM CD8+ T cells activated for 2 hr with a-CD3 and a-CD28 mAb loaded beads only or similarly activated in presence of nocodazole (10 mM). Bar graph shows AKT-Ser473 phosphorylation normalized to actin (n = 3 independent experiments). (H) Immunoblots of MEJ and PM fractions from Jurkat T cells. Blots were probed with GSK3b and COX IV antibodies (representative of n = 3 independent experiments). (I) Immunoblots of MEJ and EGC fractions from bulk CD8+ T cells either left unstimulated (–) or activated (+) with a-CD3 and a-CD28 mAb-loaded beads for 2 hr. Blots were probed for total GSK3b, pGSK3b Ser9, and COX IV (representative of n = 3 donors). (J) Left, immunoblot analysis of WT and rictor KO memory OT-I cells activated as in (D). MEJ fractions were probed for Gsk-3b, pGsk-3b Ser9, and Cox IV. Right, bar graphs display fold change in phosphoprotein levels relative to non-activated controls (n = 3 independent experiments). Data are presented as mean ± SEM. Two-tailed paired Student’s t test (D–G, J) were used to compare groups. *p < 0.05, **p < 0.01, ns, not significant. See also Figure S3.

Article Snippet: Membranes were probed with antibodies against ACC, rictor, mTOR, HK-I, HK-II, Sin1, Cox iv, Akt, pAkt Thr308, pAkt Ser473, p-(S/T) Akt substrate motif, pGsk-3b Ser9 (all from cell signaling), GRP75, VDAC1, Gsk-3b (from Abcam), actin (Sigma-Aldrich), or raptor (Bethyl).

Techniques: Western Blot, Biomarker Discovery, Control, Phospho-proteomics, Activation Assay, Two Tailed Test