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 immunolabeling: chicken-anti-GFP, # A10262 , Invitrogen (ICC 1:500) mouse-anti-Lamp1, #1D4B, Developmental Studies Hybridoma Bank* (ICC 1:100) rabbit-anti-Larp1, #PA5–62398, ThermoFisher (ICC 1:200) rabbit-anti-mTOR, #2983, Cell Signaling Technology (ICC 1:400) rabbit-anti-mTOR, #A300–503A, Bethyl Labs (ICC 1:400) rabbit-anti-Raptor, #42–4000, ThermoFisher (ICC 1:200) mouse-anti-Raptor, #ab169506, Abcam (ICC 1:200) rabbit-anti-RFP, #600–401-379, Rockland (ICC 1:500) rabbit-anti-Rictor, #2140, Cell Signaling Technology (ICC 1:200) rabbit-anti-TSC1, #PA5–20131, ThermoFisher (ICC 1:500) Isotype-specific secondary antibodies used for fluorescence imaging were Alexa fluor-conjugated and cross-absorbed (Life Technologies).

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

Journal: Scientific Reports

Article Title: DNA methylation Landscape of body size variation in sheep

doi: 10.1038/srep13950

Figure Lengend Snippet: X ray is the CpG locus, and Y ray is the DNA methylation level from 0 (no methylation) to 1(100% methylation). Region 7 is located in SMAD1 region which is on Chromosome 17 from 12,487,811 to 12,488,083. Region 9 is located in TSC1 region which is on Chromosome 3 from 3925441 to 3925953. Region 10 is located in AKT1 region which is on Chromosome 18 from 67,877,536 to 67,878,025.

Article Snippet: Separated proteins were then transferred onto nitrocellulose membranes, which were incubated overnight with one of the following primary antibodies: BMPR1B(1:1000, 40–9400, Invitrogen), TSC1(1:1000, bs-3837R, Bioss), TSC1/Ser505 (1:1000, bs-5600R, Bioss), AKT1/Thr308 (1:1000, 2965, CST), SMURF1(1:1000, bs-9391R, Bioss), SMAD1(1:1000, 6944, CST), SMAD1/Ser465(1:500, sc-101800, Santa).

Techniques: DNA Methylation Assay, Methylation

Journal: Scientific Reports

Article Title: DNA methylation Landscape of body size variation in sheep

doi: 10.1038/srep13950

Figure Lengend Snippet: ( a ) RNA expression levels of BMPR1B , SMAD1 , SUMARF1 , TSC1 , and AKT1 . Interestingly, AKT1 showed significant level in UQ compared with StH and Tan. ( b ) Protein expression levels of BMPR1B, SMAD1, TSC1 and AKT1. There is no significant protein expression level observed among breeds. ( c ) TSC1 and TSC1 (Ser505) protein expression levels among UQ, Tan, and StH.

Article Snippet: Separated proteins were then transferred onto nitrocellulose membranes, which were incubated overnight with one of the following primary antibodies: BMPR1B(1:1000, 40–9400, Invitrogen), TSC1(1:1000, bs-3837R, Bioss), TSC1/Ser505 (1:1000, bs-5600R, Bioss), AKT1/Thr308 (1:1000, 2965, CST), SMURF1(1:1000, bs-9391R, Bioss), SMAD1(1:1000, 6944, CST), SMAD1/Ser465(1:500, sc-101800, Santa).

Techniques: RNA Expression, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease

doi: 10.1016/j.jbc.2024.105641

Figure Lengend Snippet: Reagent resources and list of primers

Article Snippet: Rabbit monoclonal anti-Hamartin/TSC1 (D43E2) , Cell signaling , Cat#6935; RRID: AB_10860420.

Techniques: Electron Microscopy, Recombinant, Protease Inhibitor, Staining, Isolation, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Activation Assay, Mutagenesis

Journal: Skeletal Muscle

Article Title: Differential response of skeletal muscles to mTORC1 signaling during atrophy and hypertrophy

doi: 10.1186/2044-5040-3-6

Figure Lengend Snippet: Conditional inactivation of TSC1 in skeletal muscle. ( A ) Western blot analysis of soleus muscle from 90-day-old control (ctrl) and TSCmKO mice using antibodies directed against the proteins indicated. α-actinin is used as a loading control. ( B ) Weight of soleus (Sol), gastrocnemius (GC), plantaris (PL), tibialis anterior (TA), extensor digitorum longus (EDL) and triceps (Tri) muscles of TSCmKO and littermate control (ctrl) mice. Weight is expressed as a percentage of the weight of the same muscle in control mice after normalization to the total body weight (N = 8 to 12 mice for each genotype). Data are mean ± SEM; *** P <0.001; ** P <0.01; * P <0.05; Student’s t -test. ( C ) H&E staining of cross-sections from TA and soleus muscles of control and TSCmKO mice. Scale bar = 50 μm. ( D, E ) Fiber size distribution in soleus ( D ) and TA ( E ) muscles of 90-day-old TSCmKO and control mice (N = 4). More details of fiber size analysis are shown in Additional file : Figure S3 and in Additional file , Table S1. * P <0.05.

Article Snippet: The antibodies used were from the following sources: rabbit polyclonal antibodies directed to 4E-BP1 (Phas-I) from Zymed (Life Technologies); those recognizing Phospho-4E-BP1 (Ser65), PKB/Akt, mTOR, S6 Ribosomal Protein or Phospho-S6 Ribosomal Protein (Ser235/236) were all from Cell Signaling Technology Inc. (Danvers, MA, USA); those against FoxO1a were from Abcam plc. (Cambridge, UK); those against TSC1 were from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Western Blot, Control, Muscles, Staining

Journal: Skeletal Muscle

Article Title: Differential response of skeletal muscles to mTORC1 signaling during atrophy and hypertrophy

doi: 10.1186/2044-5040-3-6

Figure Lengend Snippet: TSC1-raptor double knockouts resemble RAmKO mice. ( A ) Western blot analysis of soleus muscles of TSCmKO, TSC-RAmKO and control (ctrl) mice using antibodies directed against the proteins indicated. An equal amount of protein was loaded in each lane. Loading control was α-actinin. ( B ) Muscle weight of the tibialis anterior (TA) and soleus (Sol) muscles of TSC-RAmKO and control mice. Muscle weight was first normalized to the body weight and is expressed as percentage of the weight of the same muscle from control mice (N = 3 mice for each genotype). ( C ) Relative mRNA expression of Pgc1α and Pgc1β in soleus muscle of TSC-RAmKO and ctrl mice. Values obtained in control mice were set to 100% (N = 3 mice). Bars in B and C represent mean ± SEM. P -values are *** P <0.001; ** P <0.01; * P <0.05. ( D ) NADH-TR staining of soleus muscle from TSC-RAmKO and control mice. Scale bar = 100 μm. ( E ) Schematic drawing of the major signaling pathways regulated by mTORC1 and their influence on protein synthesis and degradation.

Article Snippet: The antibodies used were from the following sources: rabbit polyclonal antibodies directed to 4E-BP1 (Phas-I) from Zymed (Life Technologies); those recognizing Phospho-4E-BP1 (Ser65), PKB/Akt, mTOR, S6 Ribosomal Protein or Phospho-S6 Ribosomal Protein (Ser235/236) were all from Cell Signaling Technology Inc. (Danvers, MA, USA); those against FoxO1a were from Abcam plc. (Cambridge, UK); those against TSC1 were from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Western Blot, Muscles, Control, Expressing, Staining, Protein-Protein interactions

Journal: Scientific Reports

Article Title: DNA methylation Landscape of body size variation in sheep

doi: 10.1038/srep13950

Figure Lengend Snippet: X ray is the CpG locus, and Y ray is the DNA methylation level from 0 (no methylation) to 1(100% methylation). Region 7 is located in SMAD1 region which is on Chromosome 17 from 12,487,811 to 12,488,083. Region 9 is located in TSC1 region which is on Chromosome 3 from 3925441 to 3925953. Region 10 is located in AKT1 region which is on Chromosome 18 from 67,877,536 to 67,878,025.

Article Snippet: Separated proteins were then transferred onto nitrocellulose membranes, which were incubated overnight with one of the following primary antibodies: BMPR1B(1:1000, 40–9400, Invitrogen), TSC1(1:1000, bs-3837R, Bioss), TSC1/Ser505 (1:1000, bs-5600R, Bioss), AKT1/Thr308 (1:1000, 2965, CST), SMURF1(1:1000, bs-9391R, Bioss), SMAD1(1:1000, 6944, CST), SMAD1/Ser465(1:500, sc-101800, Santa).

Techniques: DNA Methylation Assay, Methylation

Journal: Scientific Reports

Article Title: DNA methylation Landscape of body size variation in sheep

doi: 10.1038/srep13950

Figure Lengend Snippet: ( a ) RNA expression levels of BMPR1B , SMAD1 , SUMARF1 , TSC1 , and AKT1 . Interestingly, AKT1 showed significant level in UQ compared with StH and Tan. ( b ) Protein expression levels of BMPR1B, SMAD1, TSC1 and AKT1. There is no significant protein expression level observed among breeds. ( c ) TSC1 and TSC1 (Ser505) protein expression levels among UQ, Tan, and StH.

Article Snippet: Separated proteins were then transferred onto nitrocellulose membranes, which were incubated overnight with one of the following primary antibodies: BMPR1B(1:1000, 40–9400, Invitrogen), TSC1(1:1000, bs-3837R, Bioss), TSC1/Ser505 (1:1000, bs-5600R, Bioss), AKT1/Thr308 (1:1000, 2965, CST), SMURF1(1:1000, bs-9391R, Bioss), SMAD1(1:1000, 6944, CST), SMAD1/Ser465(1:500, sc-101800, Santa).

Techniques: RNA Expression, Expressing