Journal: BMC Cancer

Article Title: Up-regulation of GLI1 in vincristine-resistant rhabdomyosarcoma and Ewing sarcoma

doi: 10.1186/s12885-020-06985-0

Figure Lengend Snippet: GLI1 expression and primary cilia in RMS and EWS cell lines. a Expression of GLI1 protein by Western blot in RMS and EWS cell lines. Size markers in kilodaltons (kD) are included. GLI1 -amplified RMS-13 rhabdomyosarcoma cells or GLI1 - amplified Rh30 cells were used as positive controls. The GAPDH control was included to show comparability of protein loading among lanes. The blots have been cropped and full-length blots are presented in Supplementary Figure A. b EWS cell lines that were established following recurrence (CHLA258 and TC71) had significantly higher GLI1 expression compared with cell lines established at the time of diagnosis (CHLA9, CHLA10, and TC32) by qRT PCR. c Immunofluorescence microscopy for primary cilia in RMS and EWS cell lines. Cell line names are indicated above the images. Green = pericentrin or pericentrin2 (centriole component), red = acetylated alpha tubulin (primary cilia component), and blue = DAPI nuclear staining. Primary cilia, indicated by arrows, were seen in RD, Rh30, Rh41, CHLA9, CHLA10, CHLA258, and TC32 cells. NIH3T3 cells were used as a positive control. White dotted lines indicate cell borders. Scale bars are shown. Microsoft Exel and Adobe Photoshop were used to prepare Figure 1

Article Snippet: We incubated the cells with anti-acetylated alpha-tubulin (Sigma, St. Louis, MO, 1:5000 dilution), and pericentrin (Abcam, Cambridge, MA, 1:500 dilution) or pericentrin2 (Santa Cruz Biotech, Santa Cruz, CA, 1:500 dilution) antibodies overnight at 4 °C.

Techniques: Expressing, Western Blot, Amplification, Control, Biomarker Discovery, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining, Positive Control

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Protein expression of precipitated EVs. The serum collected from seven mice of wild-type (WT) and EML4-ALK transgenic mouse (TL group) was subjected to Western blot analysis with anti-CHL1, anti-α2 integrin, anti-β1 integrin, and anti-FGFR3 antibodies, which were cancer cell membrane BiCAT molecules previously reported. ( B ) Western blot analysis for TSG101 antigen detection in Sephacryl S-500 fractions. The fractions were concentrated with Nanosep ® centrifugal unit, and then subjected to Western blot analysis with anti-TSG101 antibody. TSG101-(Ub)n indicates ubiquitinated TSG101.

Article Snippet: After blocking with 5% skim milk solution, transferred membrane was reacted with first antibodies as follows: anti-mouse and human CHL1 antibody (AF2147 and MAB2126; R&D systems, MN; 1 μg/ml), HRP-conjugated anti-mouse and human CHL1 antibody (described above), anti-α2 integrin antibody (ab133557; Abcam; 1 μg/ml), anti-β1 integrin antibody (610467; BD transduction laboratories, NJ; 0.25 μg/ml), anti-FGFR3 antibody (c-15; Santa cruz, TX; 0.2 μg/ml 5% BSA-PBS), anti-TSG101 antibody (EXOAB-TSG101-1; System Biosciences, CA; 1:500), anti-CD63 antibody (EXOAB-CD63A-1; System Biosciences, CA; 1:500), anti-CD5L antibody (AF2834; R&D systems, MN; 0.4 μg/ml), anti-Pregnancy Zone Protein (PZP) antibody (PAG324Ra01; CLOUD-CLONE, TX; 0.5 μg/ml), anti-SLC4A1 antibody (18566-1-AP; PROTEINTECH, IL; 0.6 μg/ml), anti-Thrombospondin 1 (THBS1) antibody (PAA611Hu01; CLOUD-CLONE, TX; 0.5 μg/ml), and anti-caspase 14 antibody (MAB8215; R&D systems, MN; 0.5 μg/ml).

Techniques: Expressing, Transgenic Assay, Western Blot

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Western blot analysis of TSG101 ( A ) and CD63 ( B ) in serum EVs from WT, TL, and TS using anti-TSG101 and CD63 antibody. Arrows indicate the detected band of monomer TSG101 and CD63. The black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( C ) Western blot analysis of EMARS products with anti-CHL1 antibody. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. Arrow indicates the detected band of CHL1. Asterisk indicates unknown bands (predicted as non-specific or partial fragments).

Article Snippet: After blocking with 5% skim milk solution, transferred membrane was reacted with first antibodies as follows: anti-mouse and human CHL1 antibody (AF2147 and MAB2126; R&D systems, MN; 1 μg/ml), HRP-conjugated anti-mouse and human CHL1 antibody (described above), anti-α2 integrin antibody (ab133557; Abcam; 1 μg/ml), anti-β1 integrin antibody (610467; BD transduction laboratories, NJ; 0.25 μg/ml), anti-FGFR3 antibody (c-15; Santa cruz, TX; 0.2 μg/ml 5% BSA-PBS), anti-TSG101 antibody (EXOAB-TSG101-1; System Biosciences, CA; 1:500), anti-CD63 antibody (EXOAB-CD63A-1; System Biosciences, CA; 1:500), anti-CD5L antibody (AF2834; R&D systems, MN; 0.4 μg/ml), anti-Pregnancy Zone Protein (PZP) antibody (PAG324Ra01; CLOUD-CLONE, TX; 0.5 μg/ml), anti-SLC4A1 antibody (18566-1-AP; PROTEINTECH, IL; 0.6 μg/ml), anti-Thrombospondin 1 (THBS1) antibody (PAA611Hu01; CLOUD-CLONE, TX; 0.5 μg/ml), and anti-caspase 14 antibody (MAB8215; R&D systems, MN; 0.5 μg/ml).

Techniques: Western Blot, Molecular Weight, Purification, Immunoprecipitation, SDS Page, Fluorescence

Journal: Journal of neuropathology and experimental neurology

Article Title: The nonthiazolidinedione PPARgamma agonist L-796,449 is neuroprotective in experimental stroke.

doi: 10.1097/01.jnen.0000178852.83680.3c

Figure Lengend Snippet: FIGURE 3. L-796,449 inhibits middle cerebral artery occlusion- (MCAO) - induced inducible NO synthase and MMP-9 but not COX-2 expression after experimental stroke. Effect of L- 796,449 (1 mg/kg) on iNOS (A), COX-2 (B), and MMP-9 (C, D) after permanent MCAO. Panels (A–C) show Western blot analyses and panel (D) shows a representative zymogram from brain homogenates, and the corresponding lower panels show the densitometric analysis of bands. Data are mean 6 standard error of mean, n = 5, *, p , 0.05 versus control; #, p , 0.05 versus MCAO.

Article Snippet: Proteins were size-separated in 7% SDSpolyacrylamide gel electrophoresis (120 mA), transferred to a PVDF membrane (HybondTM-P; Amersham Biosciences Europe GmbH, Freiburg, Germany), and incubated with specific primary antibodies against iNOS (Santa Cruz Biotechnology, Santa Cruz, CA; 1:500), COX-2 (Santa Cruz, 1:1000), MMP-9 (Chemicon, Temecula, CA; 1:2000), HO-1 (Labo; 1:5000, Stressgen Biotechnologies Corp., Victoria, BC, Canada), p65 (Santa Cruz; 1:1000), and IkBa (Santa Cruz; 1:1000).

Techniques: Expressing, Western Blot, Control

Journal: The Journal of Neuroscience

Article Title: Identification of a Novel Basic Helix-Loop-Helix Gene, Heslike , and Its Role in GABAergic Neurogenesis

doi: 10.1523/JNEUROSCI.5327-03.2004

Figure Lengend Snippet: Heslike and Mash1 expression and GABAergic neurogenesis in the mesencephalon. Transverse sections of the mesencephalon were stained with antibodies. A–C, At E9.5, GABAergic neurons (GABA+GAD65+) are not yet formed (A, B), although neurons (TuJ1+) are differentiated (A). A low level of Heslike expression occurs around the alar-basal boundary (B, arrowhead). Many Heslike+ cells coexpress Mash1 (C, arrowhead, inset). D–F, D'–F', At E10.5, GABAergic neurons (GABA+GAD65+) are formed in the mantle layer just outside the Heslike+ ventricular zone (D, D', E, E'). GABA signal is also detected at the ventricular surface of the zone of GABAergic neurons (D'). Heslike+ cells coexpress Mash1 (F, F'). G–I, G'–I', At E11.5, the region for GABAergic neuron formation (GABA+GAD65+) is expanded dorsally (G, G', H, H'). This expansion follows dorsal expansion of Heslike+ domain (H, H'). Most Heslike+ cells coexpress Mash1 (I, I'). J–L, J'–L', At E12.5, after the dorsal expansion of Heslike+ domain, the region for GABAergic neuron formation (GABA+GAD65+) is further expanded dorsally (J, J', K, K'). Most Heslike+ cells coexpress Mash1 (L, L'). A higher magnification of the indicated region in D–L is shown in D'–L', respectively. Scale bars, 200 μm.

Article Snippet: The following antibodies were used at the indicated dilutions: antiphosphorylated histone 3 (Sigma, St. Louis, MO; 1:500), anti-Ki67 (BD PharMingen, San Diego, CA; 1:500), anti-β-Tubulin III (TuJ1) (Babco, Richmond, CA; 1:500), anti-glutamic acid decarboxylase 65 (GAD65) (BD PharMingen; 1:1000), anti-Mash1 (BD PharMingen, 1:1000), anti-GABA (Sigma; 1:2000), anti-Nkx2.2 (Developmental Studies Hybridoma Bank, University of Iowa, IA; 1:200), anti-Pax6 (Developmental Studies Hybridoma Bank; 1:200), anti-Shh (Developmental Studies Hybridoma Bank; 1:100), and anti-green fluorescent protein (GFP) conjugated with Alexa-488 (Molucular Probes, Eugene, OR; 1:500).

Techniques: Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: Identification of a Novel Basic Helix-Loop-Helix Gene, Heslike , and Its Role in GABAergic Neurogenesis

doi: 10.1523/JNEUROSCI.5327-03.2004

Figure Lengend Snippet: Heslike and Mash1 expression and GABAergic neurogenesis in the diencephalon. Transverse sections were stained with antibodies. A–D, At E10.5, GABAergic neurons (GABA+GAD65+) are formed in the mantle layer just outside the two stripes of Heslike+ ventricular regions in the pretectum (PT) (A, arrowheads, B). Neurons (TuJ1+) are differentiated widely in the diencephalon (A). Most Heslike+ cells coexpress Mash1 (C). Heslike+ regions overlap with Nkx2.2 expression domains (D). E–L, At E11.5 and E12.5, the dorsal stripe of Heslike+ ventricular region is expanded dorsally, and GABAergic neurons (GABA+GAD65+) are formed in the mantle layer just outside the Heslike+ ventricular zone (E, F, I, J). Most Heslike+ cells coexpress Mash1 (G, K), and some regions overlap with Nkx2.2 domains (H, L). M–P, At E11.5, there are GAD65+ cells just outside the Heslike+ region near the ZLI (M). Heslike is coexpressed with Mash1 (O, arrow) in a stripe just caudal to the ZLI, which expresses Shh (P). A higher magnification of the indicated region in N is shown in O. Scale bars, 200 μm.

Article Snippet: The following antibodies were used at the indicated dilutions: antiphosphorylated histone 3 (Sigma, St. Louis, MO; 1:500), anti-Ki67 (BD PharMingen, San Diego, CA; 1:500), anti-β-Tubulin III (TuJ1) (Babco, Richmond, CA; 1:500), anti-glutamic acid decarboxylase 65 (GAD65) (BD PharMingen; 1:1000), anti-Mash1 (BD PharMingen, 1:1000), anti-GABA (Sigma; 1:2000), anti-Nkx2.2 (Developmental Studies Hybridoma Bank, University of Iowa, IA; 1:200), anti-Pax6 (Developmental Studies Hybridoma Bank; 1:200), anti-Shh (Developmental Studies Hybridoma Bank; 1:100), and anti-green fluorescent protein (GFP) conjugated with Alexa-488 (Molucular Probes, Eugene, OR; 1:500).

Techniques: Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: Identification of a Novel Basic Helix-Loop-Helix Gene, Heslike , and Its Role in GABAergic Neurogenesis

doi: 10.1523/JNEUROSCI.5327-03.2004

Figure Lengend Snippet: Lack of GABAergic neurons in the mesencephalon of Mash1-null mice. A–F, A'–F', The wild-type (A, A', C, C', E, E') and Mash1-null (B, B', D, D', F, F') mice were analyzed at E11.5 by immunohistochemistry. In Mash1-null embryos, Heslike+ ventricular cells are increased in number (compare A' and C' with B' and D'). Although neurons (TuJ1+) are generated (F, F'), virtually no GABAergic neurons (GABA+ GAD65+) are formed in Mash1-null mesencephalon (D, D', F, F'), whereas many GABAergic neurons are generated in the mantle layer located outside the Heslike+ Mash1+ region of the wild type (C, C', E, E'). Scale bars, 200 μm.

Article Snippet: The following antibodies were used at the indicated dilutions: antiphosphorylated histone 3 (Sigma, St. Louis, MO; 1:500), anti-Ki67 (BD PharMingen, San Diego, CA; 1:500), anti-β-Tubulin III (TuJ1) (Babco, Richmond, CA; 1:500), anti-glutamic acid decarboxylase 65 (GAD65) (BD PharMingen; 1:1000), anti-Mash1 (BD PharMingen, 1:1000), anti-GABA (Sigma; 1:2000), anti-Nkx2.2 (Developmental Studies Hybridoma Bank, University of Iowa, IA; 1:200), anti-Pax6 (Developmental Studies Hybridoma Bank; 1:200), anti-Shh (Developmental Studies Hybridoma Bank; 1:100), and anti-green fluorescent protein (GFP) conjugated with Alexa-488 (Molucular Probes, Eugene, OR; 1:500).

Techniques: Immunohistochemistry, Generated

Journal: The Journal of Neuroscience

Article Title: Identification of a Novel Basic Helix-Loop-Helix Gene, Heslike , and Its Role in GABAergic Neurogenesis

doi: 10.1523/JNEUROSCI.5327-03.2004

Figure Lengend Snippet: Promotion of GABAergic neurogenesis in neural precursor cell culture by coexpression of Heslike and Mash1. A, The expression vectors for Heslike and Mash1 were cotransfected into neural precursor cells. There are two transfected cells (GFP+) in this panel. One becomes a GABAergic neuron (arrowhead; GABA+TuJ1+), whereas the other is a non-GABAergic neuron (arrow; GABA-TuJ1+). Scale bar, 20 μm. B, Quantification of GABAergic neurons. Ratios of GABAergic neurons (GABA+) per transfected cells (GFP+) are calculated. Each value with an SE represents four independent experiments performed in duplicate. Coexpression of Heslike and Mash1 promotes generation of GABAergic neurons.

Article Snippet: The following antibodies were used at the indicated dilutions: antiphosphorylated histone 3 (Sigma, St. Louis, MO; 1:500), anti-Ki67 (BD PharMingen, San Diego, CA; 1:500), anti-β-Tubulin III (TuJ1) (Babco, Richmond, CA; 1:500), anti-glutamic acid decarboxylase 65 (GAD65) (BD PharMingen; 1:1000), anti-Mash1 (BD PharMingen, 1:1000), anti-GABA (Sigma; 1:2000), anti-Nkx2.2 (Developmental Studies Hybridoma Bank, University of Iowa, IA; 1:200), anti-Pax6 (Developmental Studies Hybridoma Bank; 1:200), anti-Shh (Developmental Studies Hybridoma Bank; 1:100), and anti-green fluorescent protein (GFP) conjugated with Alexa-488 (Molucular Probes, Eugene, OR; 1:500).

Techniques: Cell Culture, Expressing, Transfection