Journal: RNA Biology

Article Title: Role of PIWI-like 4 in modulating neuronal differentiation from human embryonal carcinoma cells

doi: 10.1080/15476286.2020.1757896

Figure Lengend Snippet: Primer sequences.

Article Snippet: The blots were blocked with 5% skimmed milk for 1 hour at room temperature followed by incubation with the following primary antibodies overnight at 4°C: PIWIL1 (1:1000; ab12337, Abcam), PIWIL2 (1:1000; ab181340, Abcam), PIWIL3 (1:1000; SAB4200150, Sigma Aldrich), PIWIL4 (1:1000; ab87939, Abcam), OCT4 (1:1000; #2750, Cell Signalling Technology), NANOG (1:1000; #3580, Cell Signalling Technology), HOXA1(1:1000; ab168179, Abcam), MAP2 (1:1000; #4542, Cell Signalling Technology), TUBB3 (1:1000; #4466, Cell Signalling Technology), NLGN3 (1:400; NBP1-90,080, Novus biologicals), PTN (1:300; sc-74,443, Santa Cruz), UTX (1:1000; #33,510, Cell Signalling Technology), HA-Tag (1:1000; #3724, Cell Signalling Technology) and β-actin (1:2000; sc-47,778, Santa Cruz).

Techniques: Sequencing

Journal: RNA Biology

Article Title: Role of PIWI-like 4 in modulating neuronal differentiation from human embryonal carcinoma cells

doi: 10.1080/15476286.2020.1757896

Figure Lengend Snippet: ChIP-qPCR primer sequences.

Article Snippet: The blots were blocked with 5% skimmed milk for 1 hour at room temperature followed by incubation with the following primary antibodies overnight at 4°C: PIWIL1 (1:1000; ab12337, Abcam), PIWIL2 (1:1000; ab181340, Abcam), PIWIL3 (1:1000; SAB4200150, Sigma Aldrich), PIWIL4 (1:1000; ab87939, Abcam), OCT4 (1:1000; #2750, Cell Signalling Technology), NANOG (1:1000; #3580, Cell Signalling Technology), HOXA1(1:1000; ab168179, Abcam), MAP2 (1:1000; #4542, Cell Signalling Technology), TUBB3 (1:1000; #4466, Cell Signalling Technology), NLGN3 (1:400; NBP1-90,080, Novus biologicals), PTN (1:300; sc-74,443, Santa Cruz), UTX (1:1000; #33,510, Cell Signalling Technology), HA-Tag (1:1000; #3724, Cell Signalling Technology) and β-actin (1:2000; sc-47,778, Santa Cruz).

Techniques: Sequencing

Journal: RNA Biology

Article Title: Role of PIWI-like 4 in modulating neuronal differentiation from human embryonal carcinoma cells

doi: 10.1080/15476286.2020.1757896

Figure Lengend Snippet: PIWIL4 depletion decreased the expression of critical factors for glioma. Expression of PTN and NLGN3 during RA-mediated neuronal differentiation was profiled at the transcript level by qPCR (A) and at the protein level by immunoblotting (B). dRA: days of RA treatment. (C) Immunoblotting of PTN and NLGN3 in PIWIL4-suppressed NT2 cells which were treated with RA for 9 days. The occupancy of H3K27me3 (H3) at the promoters of PTN and NLGN3 were assessed by ChIP assays followed by PCR amplification (D) and (E) qPCR measurement. In: Input; Ig: IgG in (D). The chromatin binding of H3K27me3 was normalized to Input. pr: promoter. Bars: mean±sd; n = 4; **p < 0.01 by Student’s t-test.

Article Snippet: The blots were blocked with 5% skimmed milk for 1 hour at room temperature followed by incubation with the following primary antibodies overnight at 4°C: PIWIL1 (1:1000; ab12337, Abcam), PIWIL2 (1:1000; ab181340, Abcam), PIWIL3 (1:1000; SAB4200150, Sigma Aldrich), PIWIL4 (1:1000; ab87939, Abcam), OCT4 (1:1000; #2750, Cell Signalling Technology), NANOG (1:1000; #3580, Cell Signalling Technology), HOXA1(1:1000; ab168179, Abcam), MAP2 (1:1000; #4542, Cell Signalling Technology), TUBB3 (1:1000; #4466, Cell Signalling Technology), NLGN3 (1:400; NBP1-90,080, Novus biologicals), PTN (1:300; sc-74,443, Santa Cruz), UTX (1:1000; #33,510, Cell Signalling Technology), HA-Tag (1:1000; #3724, Cell Signalling Technology) and β-actin (1:2000; sc-47,778, Santa Cruz).

Techniques: Expressing, Western Blot, Amplification, Binding Assay

Journal: RNA Biology

Article Title: Role of PIWI-like 4 in modulating neuronal differentiation from human embryonal carcinoma cells

doi: 10.1080/15476286.2020.1757896

Figure Lengend Snippet: PIWIL4 depletion reduced the proliferation of glioma cells. (A) U87 MG glioma cells were treated for either 2 days or 3 days with the conditioned medium (CM) collected from indicated NT2 cells and subject to MTS viability assays. The gel strips above the bar graph indicate the immunoblotting of NLGN3 and PTN in the CM collected from the numbered samples. – CM: no CM used. (B) NT2 cells were treated with RA and transfected with p-siPL4 together with the plasmid overexpressing either PTN or NLGN3. The CM was then collected and used to treat U87 MG cells for 2 days. The proliferation was assessed by MTS viability assays. Bars: mean±sd; n = 3; **p < 0.01 by Student’s t-test.

Article Snippet: The blots were blocked with 5% skimmed milk for 1 hour at room temperature followed by incubation with the following primary antibodies overnight at 4°C: PIWIL1 (1:1000; ab12337, Abcam), PIWIL2 (1:1000; ab181340, Abcam), PIWIL3 (1:1000; SAB4200150, Sigma Aldrich), PIWIL4 (1:1000; ab87939, Abcam), OCT4 (1:1000; #2750, Cell Signalling Technology), NANOG (1:1000; #3580, Cell Signalling Technology), HOXA1(1:1000; ab168179, Abcam), MAP2 (1:1000; #4542, Cell Signalling Technology), TUBB3 (1:1000; #4466, Cell Signalling Technology), NLGN3 (1:400; NBP1-90,080, Novus biologicals), PTN (1:300; sc-74,443, Santa Cruz), UTX (1:1000; #33,510, Cell Signalling Technology), HA-Tag (1:1000; #3724, Cell Signalling Technology) and β-actin (1:2000; sc-47,778, Santa Cruz).

Techniques: Western Blot, Transfection, Plasmid Preparation

Journal: Experimental Brain Research

Article Title: Morc1 as a potential new target gene in mood regulation: when and where to find in the brain

doi: 10.1007/s00221-021-06171-z

Figure Lengend Snippet: Relative Morc1 mRNA expression in the rat brain. Values are given as percent expression related to the mean expression of P62 mPFC. The mean value of the three investigated animals per age/region is given as Morc1 expression in % as well as the standard deviation (SD). A Morc1 expression in different developmental stages. E: embryonic stage, P: postnatal day. B Morc1 expression in different brain regions in adult male rats. NAc nucleus accumbens, mPFC medial prefrontal cortex

Article Snippet: Fig. 1 Western blot imaging of MORC1 protein [anti-MORC1 from rabbit (1:500; 14,080–1-AP, Proteintech)] in rat brain at the age of E14 (pooled from 5 embryos), P2 (pooled from 4 pups), P22, P42, adult (P > 60), and protein from an adult rat testis (1 animal each).

Techniques: Expressing, Standard Deviation

Journal: Experimental Brain Research

Article Title: Morc1 as a potential new target gene in mood regulation: when and where to find in the brain

doi: 10.1007/s00221-021-06171-z

Figure Lengend Snippet: Western blot imaging of MORC1 protein [anti-MORC1 from rabbit (1:500; 14,080–1-AP, Proteintech)] in rat brain at the age of E14 (pooled from 5 embryos), P2 (pooled from 4 pups), P22, P42, adult (P > 60), and protein from an adult rat testis (1 animal each). Moreover, MORC1 protein was stained in one adult female C57BL/6 N Morc1 (−/−) brain using 50 µg and 100 µg protein. Imaging was performed with the ChemiDoc™ MP Imaging System (BIO-RAD). A prominent band was detected at 110 kDa (red arrow) in all rat samples but not the Morc1 (−/−) sample. Beta-actin staining revealed prominent bands at 45 kDa (ßActin) in all samples

Article Snippet: Fig. 1 Western blot imaging of MORC1 protein [anti-MORC1 from rabbit (1:500; 14,080–1-AP, Proteintech)] in rat brain at the age of E14 (pooled from 5 embryos), P2 (pooled from 4 pups), P22, P42, adult (P > 60), and protein from an adult rat testis (1 animal each).

Techniques: Western Blot, Imaging, Staining

Journal: European journal of pharmacology

Article Title: Fabry disease-associated globotriaosylceramide induces mechanical allodynia via activation of signaling through proNGF-p75 NTR but not mature NGF-TrkA.

doi: 10.1016/j.ejphar.2021.173882

Figure Lengend Snippet: Fig. 2. Signaling through proNGF–p75NTR, but not mNGF–TrkA, is required for Gb3- induced mechanical sensitization. (A) Experimental timeline of the von Frey tests and the i.pl. injection of Gb3 and anti body/antiserum or antibody/antiserum alone. (B, C) The antiserum (1 μl/paw, i.pl.) containing anti-proNGF antibody effectively recovered the Gb3-induced decrease of the 50% paw withdrawal threshold (PWT; 1 h: H = 9.40, P = 0.009; 3 h: H = 9.13, P = 0.010; 6 h: H = 6.79, P = 0.034; 24 h: H = 6.24, P = 0.044) and its area under the curve (AUC; H = 10.1, P = 0.006). n = 8 per group. The injection volume was 10 μl. (D, E) The anti-NGF antibody (1 μg/paw and 10 μg/ paw, i.pl.) failed to significantly alleviate reductions in the pain threshold and its AUC (H = 10.5, P = 0.015) in Gb3-injected mice. Vehicle, n = 7; Gb3 + Control IgG, n = 8; Gb3 + Anti-NGF antibody (1 μg), n = 9; Gb3 + Anti-NGF antibody (10 μg), n = 6. The injection volume was 10 or 20 μl. (F, G) Administration of an anti-p75NTR antibody (1 μg/paw, i.pl.) almost completely reversed the Gb3-induced decline of the 50% PWT (24 h: H = 8.44, P = 0.015) and its AUC (H = 9.16, P = 0.010) to the level of the vehicle control. Vehicle, n = 11; Gb3 + Control IgG, n = 15; Gb3 + Anti-p75NTR antibody, n = 14. The injection volume was 10 or 20 μl. (H, I) Inhibition of TrkA activity with the neutral izing antibody (1 μg/paw and 10 μg/paw, i. pl.) gave minimal influence on the Gb3- induced mechanical allodynia (AUC: H = 9.80, P = 0.020). Vehicle, n = 11; Gb3 + Control IgG, n = 11; Gb3 + Anti-TrkA anti body (1 μg), n = 7; Gb3 + Anti-TrkA anti body (10 μg), n = 7. The injection volume was 20 μl. *P < 0.05, **P < 0.01 vs the Vehicle group, #P < 0.05, ##P < 0.01 vs the Gb3 + Normal serum or Control IgG group by Dunn’s test with Holm adjustment.

Article Snippet: The details of the NGF-related antibodies/antiserum and their control IgG/serum were as follows: mouse monoclonal anti-NGF antibody (L148M, Exalpha Biologicals, Shirley, MA, USA; 1 μg and 10 μg, Shutov et al., 2016); sheep polyclonal anti-proNGF antibody (S-080-100, Biosensis, Thebarton, Australia; 1 μl J. Sugimoto et al. European Journal of Pharmacology 895 (2021) 173882 antiserum); mouse monoclonal anti-TrkA antibody (Ab00439-1.4, Absolute Antibody, Redcar, UK; 1 μg and 10 μg, Ugolini et al., 2007); rabbit polyclonal anti-p75NTR antibody (AB-N01AP, Advanced Targeting Systems, San Diego, CA, USA; 1 μg, Watanabe et al., 2008); normal mouse IgG as a control of anti-NGF or anti-TrkA antibody (0G11, Exalpha Biologicals; Ab00178-1.4, Absolute Antibody); normal rabbit IgG as a control of anti-p75NTR antibody (PM035, MBL, Nagoya, Japan); and normal sheep serum as a control of anti-proNGF antibody (S3772, Sigma-Aldrich).

Techniques: Injection, Control, Inhibition, Activity Assay

Journal: European journal of pharmacology

Article Title: Fabry disease-associated globotriaosylceramide induces mechanical allodynia via activation of signaling through proNGF-p75 NTR but not mature NGF-TrkA.

doi: 10.1016/j.ejphar.2021.173882

Figure Lengend Snippet: Fig. 3. Gb3 injection did not lead to elevation of proNGF and mNGF or their receptor expression in either plantar skin or dorsal root ganglia. There were no changes in proNGF (A, C) nor mNGF (B, D) content in the plantar skin (A, B) and dorsal root ganglia (DRG; C, D) in mice that were 1 h or 24 h post-Gb3 treatment. n = 9 per group. The injection volume was 10 or 20 μl. Gb3 injection did not modify the expression levels of p75NTR in the plantar skin (E, 1 h: n = 6 per group; 24 h: n = 7 per group) or the DRG (F, n = 7 per group). The injection volume was 10 μl. The Vehicle (Veh) group, open circles; the Gb3 group, closed circles; MW, molecular weight.

Article Snippet: The details of the NGF-related antibodies/antiserum and their control IgG/serum were as follows: mouse monoclonal anti-NGF antibody (L148M, Exalpha Biologicals, Shirley, MA, USA; 1 μg and 10 μg, Shutov et al., 2016); sheep polyclonal anti-proNGF antibody (S-080-100, Biosensis, Thebarton, Australia; 1 μl J. Sugimoto et al. European Journal of Pharmacology 895 (2021) 173882 antiserum); mouse monoclonal anti-TrkA antibody (Ab00439-1.4, Absolute Antibody, Redcar, UK; 1 μg and 10 μg, Ugolini et al., 2007); rabbit polyclonal anti-p75NTR antibody (AB-N01AP, Advanced Targeting Systems, San Diego, CA, USA; 1 μg, Watanabe et al., 2008); normal mouse IgG as a control of anti-NGF or anti-TrkA antibody (0G11, Exalpha Biologicals; Ab00178-1.4, Absolute Antibody); normal rabbit IgG as a control of anti-p75NTR antibody (PM035, MBL, Nagoya, Japan); and normal sheep serum as a control of anti-proNGF antibody (S3772, Sigma-Aldrich).

Techniques: Injection, Expressing, Molecular Weight

Journal: The American Journal of Pathology

Article Title: Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

doi: 10.1016/j.ajpath.2015.02.010

Figure Lengend Snippet: List of Antibodies Used for IHC and IF

Article Snippet: For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. shows the details of antibodies used in the study. table ft1 table-wrap mode="anchored" t5 caption a7 Name Host/isotype Source Catalog# Dilution CD326/EpCAM Mouse IgG1 Santa Cruz Biotechnology (Dallas, TX) sc-59782 1:50 K7 (cytokeratin 7) Mouse IgG1 Dako M7018 1:100 K19 (cytokeratin 19) Mouse IgG1 Abcam (Cambridge, UK) ab87014 1:50 K19 (cytokeratin 19) Mouse IgG1 Dako M0888 1:100 HepPar-1 Mouse IgG1 Dako M7158 1:50 CD133/Prominin 1 Rabbit IgG Abnova (Taipei, Taiwan) {"type":"entrez-protein","attrs":{"text":"PAB12663","term_id":"1236625334","term_text":"PAB12663"}} PAB12663 1:100 CD133/PROM1 Mouse IgG1 OriGene (Unimed Scientifica, Rome, Italy) TA309943 1:50 CD90/Thy1 Rabbit IgG Abcam ab92574 1:100 CD13 Mouse IgG1 Novacastra Reagents (Leica Biosystems, Buffalo Grove, IL) NCL-CD13-304 1:100 LGR5 Goat IgG Santa Cruz Biotechnology SC-68580 1:50 Desmin Mouse IgG1 Dako M0760 1:100 Vimentin Mouse IgG1 Santa Cruz Biotechnology sc-32322 1:100 Nestin Mouse IgG1 Santa Cruz Biotechnology sc-23927 1:100 α-SMA Mouse IgG1 Dako M0851 1:50 S100A4 Rabbit IgG Dako A5114 1:100 SNAIL Rabbit IgG Santa Cruz Biotechnology sc-28199 1:50 TWIST Rabbit IgG Santa Cruz Biotechnology sc-15393 1:50 LGR5 Rabbit IgG OriGene TA301323 1:50 NCAM-PE Mouse IgG1 BD Pharmingen (Milan, Italy) 555.516 1:50 E-cadherin Mouse IgG1 Santa Cruz Biotechnology sc-21791 1:50 P-cadherin Rabbit IgG Santa Cruz Biotechnology sc-7893 1:50 GFAP Mouse IgG1 Dako M0761 1:50 CD163 Mouse IgG1 OriGene TA506382 1:50 CD31 Mouse IgG1 Dako M0823 1:50 CD13 Mouse IgG1 Abcam Ab7417 1:100 SDF1 Rabbit IgG Santa Cruz Biotechnology sc-28876 1:50 FAP Mouse IgG1 Santa Cruz Biotechnology sc-65398 1:50 Periostin Goat IgG Santa Cruz Biotechnology sc-49480 1:50 Periostin Rabbit IgG Santa Cruz Biotechnology sc-67233 1:50 CD90-FITC Human Miltenyi Biotec (Cologne, Germany) 130-095-403 1:10 CD326 (EPCAM)-FITC Human Miltenyi Biotec 130-080-301 1:10 Goat anti-rabbit FITC IgG Abcam ab-6717 1:400 Goat anti-rabbit TRITC IgG Abcam ab-6718 1:400 Goat anti-mouse FITC IgG Abcam ab-6785 1:400 Goat anti-mouse TRITC IgG Abcam ab-6786 1:400 Open in a separate window List of Antibodies Used for IHC and IF Sections were examined in a coded fashion by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany) equipped with a Jenoptik ProgRes C10 Plus Videocam (Jenoptik, Jena, Germany).

Techniques:

Journal: FEBS Open Bio

Article Title: ProNGF processing in adult rat tissues and bioactivity of NGF prodomain peptides

doi: 10.1002/2211-5463.13768

Figure Lengend Snippet: Commercial antibodies for NGF and proNGF detection.

Article Snippet: Antibodies against proNGF (Alomone Labs, Jerusalem, Israel ANT‐005; 1 : 1000, and Biosensis, Thebarton, South Australia M‐1778‐B; 1 : 1000, S‐080; 1 : 1000), and NGF (Abcam, Cambridge, UK, ab6199, 1 : 1000) were preliminary used to evaluate their reactivity and selectivity to recognise NGF prodomain and/or its mature form by dot blot.

Techniques:

Journal: FEBS Open Bio

Article Title: ProNGF processing in adult rat tissues and bioactivity of NGF prodomain peptides

doi: 10.1002/2211-5463.13768

Figure Lengend Snippet: (A–C) Dot blot and western blot for commercial antibody selectivity towards proNGF and/or NGF. (A) Detection of NGF, proNGF, C1 and C6 by using the different antibodies against NGF and proNGF: Abcam ab6199, Alomone ANT‐005 and Biosensis M‐1778‐B or S‐080. (B, C) The representative western blots show the ability and selectivity of (B) Abcam anti‐NGF antibody and (C) Biosensis (M‐1778‐B) anti‐proNGF antibody to detect recombinant NGF and/or proNGF.

Article Snippet: Antibodies against proNGF (Alomone Labs, Jerusalem, Israel ANT‐005; 1 : 1000, and Biosensis, Thebarton, South Australia M‐1778‐B; 1 : 1000, S‐080; 1 : 1000), and NGF (Abcam, Cambridge, UK, ab6199, 1 : 1000) were preliminary used to evaluate their reactivity and selectivity to recognise NGF prodomain and/or its mature form by dot blot.

Techniques: Dot Blot, Western Blot, Recombinant

Journal: FEBS Open Bio

Article Title: ProNGF processing in adult rat tissues and bioactivity of NGF prodomain peptides

doi: 10.1002/2211-5463.13768

Figure Lengend Snippet: Expression of proNGF forms in different organs of adult male rats detected by Biosensis Antibody M‐1778‐B. Representative western blot of proNGF (Biosensis M‐1778‐B) detection in rat cortex, spinal cord and several peripheral tissues. “M” stands for protein marker.

Article Snippet: Antibodies against proNGF (Alomone Labs, Jerusalem, Israel ANT‐005; 1 : 1000, and Biosensis, Thebarton, South Australia M‐1778‐B; 1 : 1000, S‐080; 1 : 1000), and NGF (Abcam, Cambridge, UK, ab6199, 1 : 1000) were preliminary used to evaluate their reactivity and selectivity to recognise NGF prodomain and/or its mature form by dot blot.

Techniques: Expressing, Western Blot, Marker