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: bioRxiv

Article Title: mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis

doi: 10.1101/812990

Figure Lengend Snippet: A) Levels of mTOR pathway proteins in total homogenates and LD isolations at 6 hours of OA treatment. Active mTOR, Raptor, Rheb and RagA, B and C, but not Rictor or TSC1, accumulate in LDs after lysosomal inhibition. B-C) mTOR (magenta) recruitment to LDs (green) is enhanced after blocking autophagy in the OA-treated primary hepatocytes (B, quantified in C). D-E) mTOR recruitment is also enhanced after blocking autophagy through Atg7 silencing in OA-treated NIH-3T3 cells. (D quantified in E). Scale bar: 20μm. Bars are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (differences caused by lysosomal inhibitors treatment), # p<0.05, ## p<0.01, and ### p<0.001 (differences caused by treatment)

Article Snippet: Antibodies for Atg16l (PM040) from MBL; Atg7 (2631), Beclin 1 (3495), FIP200 (12436), LC3B (2775), mTOR (2983), phopho-mTOR (5536), Rag A (4357), Rag C (3360), Raptor (2280), Rictor (2114), S6 (2217), phopho-S6 (4858), TSC1 (6935) and anti-rabbit (7074) were from Cell Signaling Technology; RagB (NBP1-85801) from Novus Biologicals; Plin3 (3883,for WB) from ProSci; LAMP1 (1D4B, for WB) from Developmental Studies Hybridoma Bank; PLIN3 (GP30 for CoIP) from ProGen Biotechnik; β-actin (A5441) and anti-mouse (AP130P) and anti-rat (AP136P) from SIGMA; GAPDH (ab8245), Lamp1 (ab24170 for IF) and normal IgG (ab188776) from Abcam; Fluorescence secondary antibodies (Alexa Fluor 488 and/or Alexa Fluor 647 conjugated) and anti-guinea pig (A18775) from Fisher Scientific and Beclin 1-HRP(sc-48341 HRP) and Plin 3-HRP (sc-390968 HRP) form Santa Cruz Biotechnology.

Techniques: Inhibition, Blocking Assay

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: Molecular Therapy. Nucleic Acids

Article Title: Inflammatory-miR-301a circuitry drives mTOR and Stat3-dependent PSC activation in chronic pancreatitis and PanIN

doi: 10.1016/j.omtn.2022.01.011

Figure Lengend Snippet: Inhibition of PanIN formation in miR-301a −/− ; Pdx1-Cre ; Kras G12D mice correlates with elevated Gadd45g expression and reduced PSCs proliferation (A) Venn diagram of specific genes between predicted miR-301a targets from miRWalk database and DEGs identified by RNA-seq. (B) Real-time PCR validation of 17 significantly upregulated genes from (A). (C) Prediction of major interference sites between miR-301a and the Gadd45g mRNA 3′ UTR using TargetScan. (D) Luciferase activity in 293T cells transfected with the indicated luciferase reporter with either a control plasmid or a precursor miR-301a plasmid. (E) Western blot analysis of Gadd45g expression in 293T cells with the indicated luciferase reporter with either a control plasmid or a precursor miR-301a plasmid. (F) Western blot analysis of Gadd45g and Tsc1 expression in human PSCs transfected with anti-control (Anti-Ctl) or LNA-anti-miR-301a (Anti-miR-301a) oligonucleotide. (G) Western blot analysis of Gadd45g and Tsc1 expression in human PSCs transfected with anti-control (Anti-Ctl) or LNA-anti-miR-301a (Anti-miR-301a) oligonucleotide. (H) Expression of miR-301a in hPSCs treated with IL-6 or TGF-β. (I) Expression of miR-301a in mouse embryonic fibroblasts (MEFs), mouse PSCs (mPSCs), or Pan02 cells treated with IL-6 or TGF-β. (J) Cell viability of WT and miR-301a −/− PSCs treated with TGF-β. (K) Cell viability of miR-301a −/− PSCs transfected with shRNA-control (shCtl) or shRNA-Gadd45g (shGadd45g) and treated with TGF-β at the indicated time. Values are mean ± SD. ∗∗p < 0.01, ∗p < 0.05 indicate significant difference between the indicated groups. NS, not significant.

Article Snippet: The primary antibodies used were Tsc1 (BA2879, BOSTER), phospho-mTOR-S2448 (381557, ZENBIO), Fibronectin (ab2413, Abcam), α-SMA (ab5694, Abcam), Gadd45g (A10286, Abclonal), Stat3 (9139, CST), p-Stat3-Tyr705 (9145, CST), β-actin (AC026, Abclonal).

Techniques: Inhibition, Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Biomarker Discovery, Luciferase, Activity Assay, Transfection, Control, Plasmid Preparation, Western Blot, shRNA