Journal: Nature

Article Title: Subcellular transcriptomes and proteomes of developing axon projections in cerebral cortex

doi: 10.1038/s41586-018-0847-y

Figure Lengend Snippet: a, Biochemical analysis of GC enrichment of mTOR pathway proteins and controls, shown in triplicate western blots of homogenate (input) and GC fraction (GC fr.) pairs, derived from six independent preps. GC marker GAP43 is positive control for enrichment, Golgi marker Gm130 is negative control. b, Quantification of GC enrichment blots in a expressed as ratios of GC fr. signal over input signal, normalized to the corresponding GAP43 ratio (marked by horizontal line). Error bars indicate SEM, n≥3 litters. TSC1, Rictor, and Lamp1 are present in GCs comparable to actin and tubulin, while mTOR, LARP1, and Raptor display high GC enrichment comparable to GC marker GAP43. c, Closeups of GCs from callosal projection neurons immunostained for endogenous mTOR pathway proteins (red in overlays, heat mapped in underlying panels). Five example GCs are shown per sample to capture the representative range. Neurons were labeled via in utero electroporation at E15 with membrane-GFP (green in overlays, outlined in underlying panels), cultured at P0, fixed and stained at DIV 3. mTOR, LARP1, TSC1, and Raptor (mTORC1 marker) appear in dense local foci within GCs. Rictor (mTORC2 marker) and Lamp1 (lysosome marker) appear in fine granules distinct from GC foci. Bar (lower right) indicates heat-map color range, as well as 10 µm scale.

Article Snippet: We used the following antibodies for immunoblotting: mouse-anti-beta-actin, #A5441, Sigma (WB 1:2000) mouse-anti-GAP43, #MAB347, Chemicon (WB 1:2000) mouse-anti-GM130, #610823, BD Biosciences (WB 1:3000) mouse-anti-Lamp1, #1D4B, Developmental Studies Hybridoma Bank* (WB 1:500) rabbit-anti-Larp1, #PA5–62398, ThermoFisher (WB 1:1000) mouse-anti-MAP2, #M1406, Sigma (WB 1:1000) rabbit-anti-mTOR, #2983, Cell Signaling Technology (WB 1:1000) rabbit-anti-mTOR, #A300–504A, Bethyl Labs (WB 1:500) rabbit-anti-Raptor, #42–4000, ThermoFisher (WB 1:1000) rabbit-anti-Rictor, #2140, Cell Signaling Technology (WB 1:1000) rabbit-anti-TSC1, #PA5–20131, ThermoFisher (WB 1:1000) mouse-anti-tubulin, #MMS-435P, Covance (WB 1:2000) Isotype-specific secondary antibodies used for ECL imaging were HRP-conjugated and cross-adsorbed (Life Technologies; Abcam).

Techniques: Western Blot, Derivative Assay, Marker, Positive Control, Negative Control, Labeling, In Utero, Electroporation, Cell Culture, Staining

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