Journal: Journal of neurochemistry

Article Title: Reduced Sterol Regulatory Element-Binding Protein (SREBP) Processing Through Site-1 Protease (S1P) inhibition Alters Oligodendrocyte Differentiation in vitro

doi: 10.1111/jnc.13721

Figure Lengend Snippet: List of antibodies and primers

Article Snippet: Secondary antibodies of appropriate species and isotype used for external and internal antigens were purchased from Jackson Immunoresearch, West Grove, PA. Coverslips were mounted onto glass slides in DAPI-containing Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Host Use and Dilution Source 04 Mouse IHC 1:4 Hybridoma, Sommer and Schachner, 1981 GalC Mouse IHC 1:3 WB 1:500 Hybridoma, Ranscht et al., 1981 Olig2 Rabbit IHC 1:700 Chemicon PLP Rat IHC undiluted WB 1:1000 AA3, Yamaguchi et al., 1996 SREBP1 Rabbit IHC 1:150 WB 1:400 Santa Cruz Biotechnology SREBP2 Rabbit IHC 1:200 WB 1:150 Abcam MBP Mouse WB 1:1000 BioLegend Olig1 Mouse WB 1:1000 Millipore Sox 10 Goat WB 1:1000 Santa Cruz Biotechnology FASN Rabbit WB 1:1000 Cell Signaling ACC Rabbit WB 1:1000 Cell Signaling HGMCR Rabbit WB 1:1000 Santa Cruz Biotechnology Phospho-Tyr-530-Fyn Rabbit WB 1:600 Sigma-Aldrich Fyn Rabbit WB 1:1000 Sigma-Aldrich tubulin Mouse WB 1:10,000 Sigma-Aldrich GAPDH Mouse WB 1:6000 Millipore Open in a separate window Target Forward Sequence Reverse Sequence SREBP1 5′-CCTGCTTGGCTCTTCTCTTT-3′ 5′-CTGGTGCAGCTTATGGTAGAC-3′ SREBP2 5′-GAGGCGGACAACACACAATA-3′ 5′-CGG-CTCAGAGTCAATGGAATAG-3′ FASN 5′-GCTGCGGAAACTTCAGGAAAT-3′ 5′-AGAGACGTGTCACTCCTGGACTT-3′ ACC 5′-CTTGTGGAATGCCTTGTGATTG-3 5′-CTGCTGCCGTCATAAGACAA-3′ HMGCR 5′-CTTGTGGAATGCCTTGTGATTG-3′ 5′-AGCCGAAGCAGCACAT-GAT-3′ MBP 5′-TGGTACACACTAACCTCGGTGGAA-3′ 5′-AGCGACTCGATTCAGTGACAGGAA-3′ PKG1 5′-ATGCAAAGACTGGCCAAGCTAC-3′ 5′-AGCCA-CAGCCTCAGCATATTTC-3′ Open in a separate window List of antibodies and primers For phalloidin staining, cells were first immunolabeled with anti-O4 as described above, then fixed in 4% paraformaldehyde/4% sucrose, washed in PBS, permeablized in 0.5% Triton X-100 followed by incubation with Phalloidin-Rhodamine (500 nM in BSA/CAS-Block; Sigma-Aldrich) for 2 hrs at RT.

Techniques:

Journal: Journal of neurochemistry

Article Title: Reduced Sterol Regulatory Element-Binding Protein (SREBP) Processing Through Site-1 Protease (S1P) inhibition Alters Oligodendrocyte Differentiation in vitro

doi: 10.1111/jnc.13721

Figure Lengend Snippet: (A, B, C) show Western blots and densitometric analysis of SREBP-1 (A) and SREBP-2 (B) protein immunoreactivities from oligodendrocyte cultures grown with or without 1 μM S1P inhibitor after 1 and 3 days in DM, with GAPDH as a loading control. Graph (C) represents the densitometric analysis of SREBP-2 mature form immunoreactivity normalized to its loading control shown in its respective Western blot at 3 days. Graph data are expressed as mean ± S.E.M. from 3 independent experiments. In (D, E), OPC and oligodendrocyte cultures grown for 1, 2 and 3 days in DM with or without S1P inhibitor (1 μM) were subjected to qRT-PCR analysis to determine SREBP-1 (D) and SREBP-2 (E) mRNA expressions relative to that of OPCs. Graph data are expressed as mean ± S.E.M. from 3 independent experiments. *p<0.01, **p<0.005, ***p<0.0001 vs. control. (F, G), show Western blot and densitometric analyses of the effect of 1μM S1P inhibitor on Olig1, Olig2 and Sox10 transcription factor immunoreactivities from cell extracts of differentiating oligodendrocytes after exposure for 1 and 3 days. Alpha-tubulin and GAPDH were used as loading controls. Representative blot data from 3–4 independent experiments.

Article Snippet: Secondary antibodies of appropriate species and isotype used for external and internal antigens were purchased from Jackson Immunoresearch, West Grove, PA. Coverslips were mounted onto glass slides in DAPI-containing Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Host Use and Dilution Source 04 Mouse IHC 1:4 Hybridoma, Sommer and Schachner, 1981 GalC Mouse IHC 1:3 WB 1:500 Hybridoma, Ranscht et al., 1981 Olig2 Rabbit IHC 1:700 Chemicon PLP Rat IHC undiluted WB 1:1000 AA3, Yamaguchi et al., 1996 SREBP1 Rabbit IHC 1:150 WB 1:400 Santa Cruz Biotechnology SREBP2 Rabbit IHC 1:200 WB 1:150 Abcam MBP Mouse WB 1:1000 BioLegend Olig1 Mouse WB 1:1000 Millipore Sox 10 Goat WB 1:1000 Santa Cruz Biotechnology FASN Rabbit WB 1:1000 Cell Signaling ACC Rabbit WB 1:1000 Cell Signaling HGMCR Rabbit WB 1:1000 Santa Cruz Biotechnology Phospho-Tyr-530-Fyn Rabbit WB 1:600 Sigma-Aldrich Fyn Rabbit WB 1:1000 Sigma-Aldrich tubulin Mouse WB 1:10,000 Sigma-Aldrich GAPDH Mouse WB 1:6000 Millipore Open in a separate window Target Forward Sequence Reverse Sequence SREBP1 5′-CCTGCTTGGCTCTTCTCTTT-3′ 5′-CTGGTGCAGCTTATGGTAGAC-3′ SREBP2 5′-GAGGCGGACAACACACAATA-3′ 5′-CGG-CTCAGAGTCAATGGAATAG-3′ FASN 5′-GCTGCGGAAACTTCAGGAAAT-3′ 5′-AGAGACGTGTCACTCCTGGACTT-3′ ACC 5′-CTTGTGGAATGCCTTGTGATTG-3 5′-CTGCTGCCGTCATAAGACAA-3′ HMGCR 5′-CTTGTGGAATGCCTTGTGATTG-3′ 5′-AGCCGAAGCAGCACAT-GAT-3′ MBP 5′-TGGTACACACTAACCTCGGTGGAA-3′ 5′-AGCGACTCGATTCAGTGACAGGAA-3′ PKG1 5′-ATGCAAAGACTGGCCAAGCTAC-3′ 5′-AGCCA-CAGCCTCAGCATATTTC-3′ Open in a separate window List of antibodies and primers For phalloidin staining, cells were first immunolabeled with anti-O4 as described above, then fixed in 4% paraformaldehyde/4% sucrose, washed in PBS, permeablized in 0.5% Triton X-100 followed by incubation with Phalloidin-Rhodamine (500 nM in BSA/CAS-Block; Sigma-Aldrich) for 2 hrs at RT.

Techniques: Western Blot, Quantitative RT-PCR

Journal: Journal of neurochemistry

Article Title: Reduced Sterol Regulatory Element-Binding Protein (SREBP) Processing Through Site-1 Protease (S1P) inhibition Alters Oligodendrocyte Differentiation in vitro

doi: 10.1111/jnc.13721

Figure Lengend Snippet: (A, B, C, D) show representative photomicrographs of oligodendrocytes labeled with anti-O4 (A, B), anti-GalC (C, D) or anti-PLP (E, F) and grown in DM for 3 days without (A, C, E) or with 1 μM S1P inhibitor (B, D, F). In controls, oligodendrocyte differentiation marker immunoreactivity is present throughout the cell including its network of processes (A, C, E), whereas in S1P inhibitor’s presence cell markers are primarily confined to the cell body (B, D, F). Corresponding DAPI staining of nuclei is shown for each panel. Scale bar: 25 μm. (G, H, I, J) show the quantification of the alteration of oligodendrocyte differentiation markers. In (G), the number of process-bearing GalC+ cells was quantified and normalized to the number of Olig2+ cells, and expressed as percent of control values. In (H, I and J), the number of “ringed” cells for each marker of differentiation (O4, GalC or PLP) was normalized to the total number of cells (ringed + process-bearing) expressing that particular marker. Data are expressed as mean ± S.E.M. from 2–4 independent experiments. *p<0.0001 vs. control.

Article Snippet: Secondary antibodies of appropriate species and isotype used for external and internal antigens were purchased from Jackson Immunoresearch, West Grove, PA. Coverslips were mounted onto glass slides in DAPI-containing Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Host Use and Dilution Source 04 Mouse IHC 1:4 Hybridoma, Sommer and Schachner, 1981 GalC Mouse IHC 1:3 WB 1:500 Hybridoma, Ranscht et al., 1981 Olig2 Rabbit IHC 1:700 Chemicon PLP Rat IHC undiluted WB 1:1000 AA3, Yamaguchi et al., 1996 SREBP1 Rabbit IHC 1:150 WB 1:400 Santa Cruz Biotechnology SREBP2 Rabbit IHC 1:200 WB 1:150 Abcam MBP Mouse WB 1:1000 BioLegend Olig1 Mouse WB 1:1000 Millipore Sox 10 Goat WB 1:1000 Santa Cruz Biotechnology FASN Rabbit WB 1:1000 Cell Signaling ACC Rabbit WB 1:1000 Cell Signaling HGMCR Rabbit WB 1:1000 Santa Cruz Biotechnology Phospho-Tyr-530-Fyn Rabbit WB 1:600 Sigma-Aldrich Fyn Rabbit WB 1:1000 Sigma-Aldrich tubulin Mouse WB 1:10,000 Sigma-Aldrich GAPDH Mouse WB 1:6000 Millipore Open in a separate window Target Forward Sequence Reverse Sequence SREBP1 5′-CCTGCTTGGCTCTTCTCTTT-3′ 5′-CTGGTGCAGCTTATGGTAGAC-3′ SREBP2 5′-GAGGCGGACAACACACAATA-3′ 5′-CGG-CTCAGAGTCAATGGAATAG-3′ FASN 5′-GCTGCGGAAACTTCAGGAAAT-3′ 5′-AGAGACGTGTCACTCCTGGACTT-3′ ACC 5′-CTTGTGGAATGCCTTGTGATTG-3 5′-CTGCTGCCGTCATAAGACAA-3′ HMGCR 5′-CTTGTGGAATGCCTTGTGATTG-3′ 5′-AGCCGAAGCAGCACAT-GAT-3′ MBP 5′-TGGTACACACTAACCTCGGTGGAA-3′ 5′-AGCGACTCGATTCAGTGACAGGAA-3′ PKG1 5′-ATGCAAAGACTGGCCAAGCTAC-3′ 5′-AGCCA-CAGCCTCAGCATATTTC-3′ Open in a separate window List of antibodies and primers For phalloidin staining, cells were first immunolabeled with anti-O4 as described above, then fixed in 4% paraformaldehyde/4% sucrose, washed in PBS, permeablized in 0.5% Triton X-100 followed by incubation with Phalloidin-Rhodamine (500 nM in BSA/CAS-Block; Sigma-Aldrich) for 2 hrs at RT.

Techniques: Labeling, Marker, Staining, Expressing