Journal: The Journal of Cell Biology

Article Title: Deletion of Murine SMN Exon 7 Directed to Skeletal Muscle Leads to Severe Muscular Dystrophy

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Figure Lengend Snippet: Analysis of SMN and components of the DGC. (A) Semiquantitative RT-PCR amplification of SMN transcripts from skeletal muscle using primers flanking exon 7 reveals transcripts containing (SMN FL) or lacking exon 7 (SMNΔ7, first panel). Primers ex5sou2 and ex7sou2 were used to amplify SMN transcripts containing exon 7 only (SMN FL, second panel). Aldolase A cDNA was coamplified and used as internal control (third panel). RT-PCR analysis reveals SMN FL transcript only in control (+/+, lane 2). In addition to the full-length RT-PCR product (SMN FL), a shorter product corresponding to SMNΔ7 transcript is detected in +/SMN Δ 7 tissue (lane 1). In mutant mice (MM, lanes 3–7), note the dramatic reduction of full-length product in skeletal muscle from 15 d of age (15 d., lane 5–7) whereas the SMN Δ 7 is the predominant form. Note the slight decrease of SMN transcript during the postnatal period of control mice (+/+, fourth panel). (B) Tissue specific deletion of the SMN F7 allele was demonstrated by PCR analysis of DNA extracted from a variety of tissues of mice carrying the ( SMN F7/ + , HSA-Cre) genotype using primers PHR5 and GS8. A 450-bp fragment was successfully amplified in skeletal muscles but not in the other tissues indicating the presence of Cre recombinase activity restricted to skeletal muscle (upper panel). PCR amplification analysis using primers flanking SMN exon 4 was used as internal positive control (630 bp, lower panel; ). (C) Western blot analysis of proteins extracted from skeletal muscle using a monoclonal antibody directed against the NH 2 terminus of SMN. Note the marked reduction of SMN levels from 15-d-old mutant mice (MM, 15 d., lane 8–10) compared with control (+/+, lanes 1–5). Incubation with monoclonal anti-actin antibody was used as internal control. (D and E) Western blot analysis of proteins extracted from skeletal muscle using a monoclonal anti-dystrophin antibody (MANDRA1, D) and polyclonal antibodies against utrophin (E). Note the reduction of dystrophin in 4-wk-old mice ( SMN F7/ Δ 7 , HSA-Cre , lanes 1–4) compared with control (+/+) associated with an upregulation of utrophin expression (E). Actin was used as internal control. Relative quantitation was achieved by densitometric scanning and the amount is indicated. MM, mutant mice; +/+, wild type; SMNFL, full-length SMN transcript; SMNΔ7, SMN transcript lacking exon 7; d., postnatal day.

Article Snippet: Polyclonal antibodies against murine collagen IV were purchased from AbCys.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Control, Mutagenesis, Muscles, Activity Assay, Positive Control, Western Blot, Incubation, Expressing, Quantitation Assay

Journal: The Journal of Cell Biology

Article Title: Deletion of Murine SMN Exon 7 Directed to Skeletal Muscle Leads to Severe Muscular Dystrophy

doi:

Figure Lengend Snippet: Immunofluorescent staining of dystrophin (A and B) and utrophin (C and D) on transverse frozen sections of skeletal muscle from control (A and C) and ( SMN F7/ Δ 7 , HSA-Cre ) mice (B and D). The MANDRA1 anti-dystrophin antibody stains the sarcolemma of muscle fibers from control mouse tissue (A) although it fails to detect the sarcolemma of some muscle fibers from mutant mice (B). In control mouse tissue, the polyclonal antiutrophin antibodies stain the neuromuscular junction only (C, arrow), although a marked extrajunctional labeling of sarcolemma is observed in mutant mice (D). Double immunostaining experiment of dystrophin (E and F) and utrophin (E′ and F′) on transverse frozen sections of skeletal muscle from control (E, E′, and E′′), and ( SMN F7/ Δ 7 , HSA-Cre ) mice (F, F′, and F′′). In mutant mouse, some muscle fibers lacking dystrophin sarcolemmal staining (F, filled arrow) display an upregulation of the sarcolemmal staining of utrophin (F′), whereas in other muscle fibers the utrophin sarcolemmal staining is observed despite the expression of dystrophin at the plasma membrane (open arrow). (E′′ and F′′) merged images. Bars: (A–D) 50 μm; (E–F′′) 35 μm.

Article Snippet: Polyclonal antibodies against murine collagen IV were purchased from AbCys.

Techniques: Staining, Control, Mutagenesis, Labeling, Double Immunostaining, Expressing, Clinical Proteomics, Membrane

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

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Figure Lengend Snippet: Primary structure of the HIV-1 Gag polyprotein proline-rich loop that binds cyclophilin A and confers cyclophilin A dependence on HIV-1 replication. The numbers refer to amino acid residues with respect to the amino terminus of the Gag polyprotein. The asterisks indicate the positions of G221, P222, and A224, residues important to this study.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques:

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

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Figure Lengend Snippet: HIV-1 A224E mutant virus is cyclosporine resistant in Jurkat cells and cyclosporine dependent in H9 cells. Infection of the Jurkat T-cell line (A) or of the H9 T-cell line (B) was initiated by transfection of proviral DNA pNL4-3 encoding the A224E amino acid mutation. Cyclosporine (CsA) was maintained in the cell culture medium at the indicated concentrations. The accumulation of extracellular virions in the culture supernatant resulting from viral spread through the culture is indicated by the reverse transcriptase (RT) activity in the supernatant at the indicated times posttransfection.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Mutagenesis, Infection, Transfection, Cell Culture, Activity Assay

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

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Figure Lengend Snippet: P222A, a Gag mutation that confers reduced cyclophilin A affinity to the Gag polyprotein, rescues the replication of HIV-1 mutant A224E in H9 cells when the two mutations are expressed in cis. Jurkat T cells (A) or H9 T cells (B) were transfected with the wild-type proviral DNA pNL4-3 or with pNL4-3 encoding the P222A/A224E double mutation. RT, reverse transcriptase.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Mutagenesis, Transfection

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: H9 cells express more cyclophilin A (CyPA) than do Jurkat cells. (A) Jurkat cells (lanes 1, 3, and 5) and H9 cells (lanes 2, 4, and 6 to 9) were grown to a density of 106/ml, lysed in radioimmunoprecipitation assay buffer, and processed for Western blotting with a primary anti-cyclophilin A antibody. Lanes 1 to 4, samples normalized by cell number. The quantity of soluble cell lysate loaded per lane, as measured by number of cell equivalents, is indicated above the lanes. Lanes 5 to 9, samples normalized by quantity of total protein in the soluble cell lysate. The numbers above the lanes indicate the amount of total protein loaded per well. (B) Replication of wild-type HIV-1 in Jurkat or H9 cells after initiation of infection with a common stock of virus produced by transfection of 293T cells with pNL4-3. RT, reverse transcriptase. (C) Virions were purified from the supernatants of the cultures used for panel B on day 8 postinfection. Virion-associated protein was probed in a Western blot with a primary anti-cyclophilin A antibody. Lanes 1 and 2, samples from Jurkat and H9 cell cultures, respectively, normalized for reverse transcriptase activity and capsid protein. Lanes 3 and 4, serial twofold dilutions of the H9 sample, as indicated.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Radio Immunoprecipitation, Western Blot, Infection, Produced, Transfection, Purification, Activity Assay

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

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Figure Lengend Snippet: HIV-1 proviruses expressing Gag mutants with attenuated cyclophilin A affinity cannot promote a spreading infection in Jurkat cells (A) but do replicate in H9 cells (B). Infection was initiated by direct transfection of wild-type or mutant proviral DNA pNL4-3 into the T-cell lines. RT, reverse transcriptase.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Expressing, Infection, Transfection, Mutagenesis

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: HIV-1 replication in H9 cells is resistant to inhibition by cyclosporine. Infection of Jurkat T cells (A) or of H9 T cells (B) was initiated by transfection of wild-type (WT) proviral DNA pNL4-3. Cyclosporine (CsA) was maintained in the cell culture medium for the duration of the experiment at the indicated concentrations. RT, reverse transcriptase.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Inhibition, Infection, Transfection, Cell Culture

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

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Figure Lengend Snippet: HIV-1 Gag mutants expressed in cis do not rescue the replication of Vif-defective HIV-1 in H9 cells. Infection of H9 T cells was initiated by transfection of wild-type (WT) or mutant proviral DNA pNL4-3. RT, reverse transcriptase. The Vif− virus is pNL4-3 with a deletion in the vif open reading frame (21).

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Infection, Transfection, Mutagenesis