Journal: Molecules

Article Title: Oridonin Induces Apoptosis in Esophageal Squamous Cell Carcinoma by Inhibiting Cytoskeletal Protein LASP1 and PDLIM1

doi: 10.3390/molecules28020805

Figure Lengend Snippet: Top Candidate Proteins Involved in Oridonin-treated ESCC.

Article Snippet: Membranes were blocked with 5% Beef Serum Albumin (BSA) and then incubated with primary antibodies: mouse Hsp70 antibody (mAb; Abcam, Boston, MA, USA), rabbit ENO1, LASP1, GAPDH antibodies (#3810 (pAb), #8636 (pAb), #2218 (mAb); cell signaling, Danvers, MA, USA), rabbit PDLIM1 antibody (ABclonal, Cambridge, MA, USA), and mouse β -Actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Techniques: Variant Assay

Journal: Molecules

Article Title: Oridonin Induces Apoptosis in Esophageal Squamous Cell Carcinoma by Inhibiting Cytoskeletal Protein LASP1 and PDLIM1

doi: 10.3390/molecules28020805

Figure Lengend Snippet: Gene Set Enrichment Analysis and western blotting assessed that oridonin inhibits LASP1 and PDLIM1 on ESCC. ( A ) Molecular function classification of the protein candidates involved in oridonin treatment. The top three are cadherin binding, cadherin binding involved in cell–cell adhesion, and heat shock protein binding. ( B ) The top three cellular component classifications are focal adhesion, cell-substrate junction, and cell cortex. ( C ) The top three biological processes are negative regulation of apoptotic signaling pathway, removal of superoxide radicals, and cellular response to oxygen radicals. ( D ) Protein expression of LASP1 and PDLIM1 under oridonin treatment decreased compared to untreated cell lysates. Hsp70 expression increased in oridonin-treated cell lysates compared to untreated while ENO1 decreased a little in treated TE-8.

Article Snippet: Membranes were blocked with 5% Beef Serum Albumin (BSA) and then incubated with primary antibodies: mouse Hsp70 antibody (mAb; Abcam, Boston, MA, USA), rabbit ENO1, LASP1, GAPDH antibodies (#3810 (pAb), #8636 (pAb), #2218 (mAb); cell signaling, Danvers, MA, USA), rabbit PDLIM1 antibody (ABclonal, Cambridge, MA, USA), and mouse β -Actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Techniques: Western Blot, Binding Assay, Protein Binding, Expressing

Journal: Molecules

Article Title: Oridonin Induces Apoptosis in Esophageal Squamous Cell Carcinoma by Inhibiting Cytoskeletal Protein LASP1 and PDLIM1

doi: 10.3390/molecules28020805

Figure Lengend Snippet: Interested Genes for Protein Assessment Involved in Oridonin Treatment.

Article Snippet: Membranes were blocked with 5% Beef Serum Albumin (BSA) and then incubated with primary antibodies: mouse Hsp70 antibody (mAb; Abcam, Boston, MA, USA), rabbit ENO1, LASP1, GAPDH antibodies (#3810 (pAb), #8636 (pAb), #2218 (mAb); cell signaling, Danvers, MA, USA), rabbit PDLIM1 antibody (ABclonal, Cambridge, MA, USA), and mouse β -Actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Techniques: Binding Assay, Protein Binding

Journal: Cells

Article Title: Calreticulin Shortage Results in Disturbance of Calcium Storage, Mitochondrial Disease, and Kidney Injury

doi: 10.3390/cells11081329

Figure Lengend Snippet: Activation of PKA and glycolysis in Calr +/− kidney. The intensive mitochondrial damage results in an energy shortage. To overcome the energy crisis, the kidney cells in Calr +/− mice activate glycolysis. ( A ): Up-regulation of Slc5a1 a sodium/glucose cotransporter 1, which actively transports glucose into the cell, and PcK1 is also up-regulated to actively augment the glucose synthesis from lactate. ( B ): Enolase 1 is also up-regulated to favor the energy production from glucose. ( C ): PKA is significantly up-regulated and activated to accelerate glycolysis and to promote energy production, and the important glycolysis kinase PFK is significantly up-regulated in Calr +/− mouse kidneys. ( D ): Parallel to the activation of glycolysis, the enzymes involved in production of the alternative energy from phosphocreatine are also up-regulated in Calr +/− mouse kidney. data (*: p < 0.05) **: p < 0.01, ***: p < 0.001).

Article Snippet: Polyclonal rabbit anti-LC3 (NB-100-2220) was from Novus Biologicals (Centennial, CO, USA); rabbit monoclonal anti-Becn-1 (11//2016) was from Cell Signaling (Frankfurt am Main, Germany); rabbit monoclonal anti-Eno1 (ab155102), anti-Prdx6 (EPR3754), anti-iNos1 (ab178945), anti-Ncx1 (ab177952), anti-Pvalb (ab181086), anti-Phb (ab75766), anti-p65 (ab32536), anti-Trpv5 (ab137028), anti-Vdac1 (ab154856), anti-Col-1 (ab270994), rabbit polyclonal anti-Bcl-2 (ab196495), goat anti-Calr (ab2907), anti-Pdia3 (ab228789), anti-pEif2-alpha (ab131505), mouse monoclonal anti-Calm1 (ab2860), anti-Canx (ab112995), and anti-IkB (ab12134) antibodies were from Abcam (Berlin, Germany).

Techniques: Activation Assay

Journal: Nature neuroscience

Article Title: A glycolytic shift in Schwann cells supports injured axons

doi: 10.1038/s41593-020-0689-4

Figure Lengend Snippet: a-i, Representative immunofluorescence for the indicated metabolic components on longitudinal frozen sections from uninjured control nerve segments and axotomized distal sciatic nerve stumps at the shown post-injury times. HK2: Hexokinase 2 (a). GPI: Glucose-6-phosphate isomerase (b). ALDA: Aldolase A (c). GAPDH: Glyceraldehyde-3-phosphate dehydrogenase (d). PGK1: Phosphoglycerate kinase 1 (e). PGAM1: Phosphoglycerate mutase 1 (f). ENO1: Enolase 1 (g). PKM1: Pyruvate kinase M1 (h). LDHB: Lactate dehydrogenase B (i). Arrows depict colocalization in SCs. Scale bars: 50μm. The experiments for each component were reproduced three times independently with similar results.

Article Snippet: Following primary antibodies were used for nerve and teased fiber immunofluorescence: Neurofilament 200 (1:500, Sigma, N4142), Hexokinase I C35C4 (1:200, Cell Signaling, 2024), Hexokinase II C64G5 (1:200, Cell Signaling, 2867), GPI (1:200, Proteintech, 15171-1-AP), PFKM (1:200, Proteintech, 55028-1-AP), Aldolase A D73H4 (1:200, Cell Signaling, 8060), GAPDH (1:500, Sigma, G9545), PGK1 (1:150, Proteintech, 17811-1-AP), PGAM1 D3J9T (1:50, Cell Signaling, 12098), Enolase-1 D2S1A (1:200, Cell Signaling, 13410), PKM1 D30G6 (1:400, Cell Signaling, 7067), PKM2 D78A4 (1:200, Cell Signaling, 4053), PFKFB3 D7H4Q (1:200, Cell Signaling, 13123), LDHA/LDHC C28H7 (1:200, Cell Signaling, 3558), LDHB (1:200, Proteintech, 14824-1-AP), Glut1 D3J3A (1:200, Cell Signaling, 12939), PDHK1 C47H1 (1:100, Cell Signaling, 3820), Pyruvate Dehydrogenase C54G1 (1:100, Cell Signaling, 3205), CS (1:150, Proteintech, 16131-1-AP), IDH3A (1:100, Proteintech, 15909-1-AP), OGDH (1:50, Proteintech, 15212-1-AP), MCT1 M-45 (1:200, Santa Cruz Biotechnology, sc-50325), MCT4 H-90 (1:250, Santa Cruz Biotechnology, sc-50329), Phospho-S6 Ribosomal Protein (Ser240/244) D68F8 (1:800, Cell Signaling, 5364), Phospho-Akt (Ser473) D9E (1:100, Cell Signaling, 4060), HIF-1 alpha (1:200, Novus Biologicals, NB100-479), c-Myc D84C12 (1:800, Cell Signaling, 5605), c-Myc/N-Myc D3N8F (1:800, Cell Signaling, 13987), Phospho-AMPKα (Thr172) 40H9 (1:100, Cell Signaling, 2535), S100 4C4.9 (1:200, Thermo Fisher Scientific, MA5-12966), P0 (1:1000, Aves Labs, PZO), TUJ1/TUBB3 (1:500, BioLegend, 801202).

Techniques: Expressing, Immunofluorescence

Journal: Molecular cell

Article Title: Imprinted maternally-expressed microRNAs antagonize paternally-driven gene programs in neurons

doi: 10.1016/j.molcel.2020.01.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Enolase-I , Cell Signaling Technology , Cat#3810; RRID: AB_2246524.

Techniques: Recombinant, Luciferase, Mutagenesis, SYBR Green Assay, Expressing, Transfection, In Utero, Electroporation, Plasmid Preparation, Software

Journal: Cell Death & Disease

Article Title: Novel ginsenoside derivative 20( S )-Rh2E2 suppresses tumor growth and metastasis in vivo and in vitro via intervention of cancer cell energy metabolism

doi: 10.1038/s41419-020-02881-4

Figure Lengend Snippet: a Both α-enolase and stathmin were suppressed after 20( S )-Rh2E2 and 20( R )-Rh2E2 treatment. Representative immunoblots and the protein quantification were shown from three independent experiments; **P < 0.01, ***P < 0.001, one-way ANOVA analysis. b Quantification of metastatic markers α-enolase and stathmin genes expression in 20( S ) / ( R )-Rh2E2-treated CCD19Lu cells by RT-qPCR. c 20( S )-Rh2E2 and 20( R )-Rh2E2 suppressed the expression of α-enolase and stathmin in tumor tissues harvested from LLC-1 xenograft mice. α-enolase and stathmin staining images were representative of 5 tumor sections from 6 animals of each group. The level of signal intensity was scored from 1–5 (5 is maximum) and took the average from five different views of each section taken in ×20 magnifications. d 20( S )-Rh2E2 and 20( R )-Rh2E2 suppressed the release of lactate in LLC-1 and H1299 cancer cells. *P < 0.05, **P < 0.01, * **P < 0.001, one-way ANOVA analysis.

Article Snippet: The following reagents from other suppliers were used: phospho-p53 kinase (Ser 15 ) rabbit mAb (CST, 9284, USA), p53 kinase rabbit mAb (CST, 9282, USA), phospho-p38 kinase (Thr 180 /Tyr 182 ) rabbit mAb (CST, 4511, USA), p38 kinase rabbit mAb (CST, 8690, USA), phospho-JNK kinase (Thr 183 /Tyr 185 ) rabbit mAb (CST, 4668, USA), JNK kinase rabbit mAb (CST, 9252, USA), phospho-ERK kinase (Thr 202 /Tyr 204 ) rabbit mAb (CST, 4370, USA), ERK kinase rabbit mAb (CST, 4695, USA), α-enolase kinase rabbit mAb (CST, 3810, USA), stathmin kinase rabbit mAb (CST, 3352, USA), phospho-AMP-activated protein kinase (AMPK; Thr 172 ) rabbit mAb (CST, 2531, USA), AMPK rabbit mAb (CST, 2532, USA), anti-β-of actin mouse monoclonal IgG1 (Santa Cruz, sc-47778, USA), rabbit anti-mouse IgG (H +L) secondary antibody TRITC (Invitrogen, PA1-28565, USA), IRDye 800CW goat anti-mouse IgG (H + L) secondary antibody (Li-COR, 926-32210, USA), IRDye 800CW goat anti-rabbit IgG (H + L) secondary antibody (Li-COR, 926–32211, USA), Compound C (Calbiochem, 171260, USA), SB203580 (CST, 5633, USA), SP600125 (CST, 8177, USA), U0126 (CST, 9903, USA), antibody against LC3-II (CST, 2775, USA).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Staining