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: 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: PLoS ONE

Article Title: Searching for Cellular Partners of Hantaviral Nonstructural Protein NSs: Y2H Screening of Mouse cDNA Library and Analysis of Cellular Interactome

doi: 10.1371/journal.pone.0034307

Figure Lengend Snippet: Intersection of closest shared TULV and PUUV NSs -linked nodes. Primary nodes are in red, secondary nodes in pink. Secondary nodes that do not directly connect to two different primary nodes are in white. Metanodes are represented by pink diamonds.

Article Snippet: Samples were incubated 1 h at 37°C with rabbit polyclonal antibodies against TULV NSs-GST fusion protein and mouse monoclonal anti-ACBD3 antibody (Abnova, Taipei, Taiwan).

Techniques:

Journal: PLoS ONE

Article Title: Searching for Cellular Partners of Hantaviral Nonstructural Protein NSs: Y2H Screening of Mouse cDNA Library and Analysis of Cellular Interactome

doi: 10.1371/journal.pone.0034307

Figure Lengend Snippet: Primary cells were infected with TULV strain Lodz for 9 days and fixed on coverslips. Cells were stained for the viral NSs protein, which was seen as bright spots around perinuclear area (A), and ACBD3 protein (B). FRET assay: D pre , donor intensity before bleaching, D post , donor intensity after bleaching, A pre , acceptor intensity before bleaching, A post , acceptor intensity after bleaching, FRET eff , calculated efficiency of FRET.

Article Snippet: Samples were incubated 1 h at 37°C with rabbit polyclonal antibodies against TULV NSs-GST fusion protein and mouse monoclonal anti-ACBD3 antibody (Abnova, Taipei, Taiwan).

Techniques: Infection, Staining

Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Involvement of HMGB1-targeted receptors in the CIPN caused by bortezomib. Bortezomib at 0.4 mg/kg or vehicle was administered i.p. on days 0, 2, 5, 7, 9, and 12. ( A , B ) Preventive ( A ) or therapeutic ( B ) effect of FPS-ZM1, TAK-242, and AMD3100, antagonists of RAGE, TLR4, and CXCR4, respectively. The mice received repeated i.p. administration of FPS-ZM1 at 1 mg/kg, TAK-242 at 3 mg/kg, AMD3100 at 8 mg/kg, or vehicle, 30 min before each dose of bortezomib ( A ), or single i.p. administration of each of them on day 14 ( B ). ( C , D ) Protein levels of RAGE, TLR4, and CXCR4 in the dorsal root ganglion ( C ) and sciatic nerves ( D ) on day 14 after the onset of bortezomib treatment. ( E , F ) Lack of preventive ( E ) and therapeutic ( F ) effects of TH1020, a TLR5 antagonist. The mice received repeated ( E ) or single ( F ) i.p. administration of TH1020 at 1 mg/kg or vehicle, in mice treated with bortezomib according to the above-mentioned schedules. Data show the mean with S.E.M for 5–6 ( A , B , F ), 5–7 ( C ), 6–7 ( D ), or 6 ( E ) mice. V, vehicle; BTZ, bortezomib; DRG, dorsal root ganglion; * p < 0.05, ** p < 0.01 vs. V ( C ) or V in V-treated mice ( A , B , E , F ). † p < 0.05, †† p < 0.01 vs. V in BTZ-treated mice.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques:

Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Scheme for bortezomib-induced caspase-dependent HMGB1 release from macrophages and CIPN development, in contrast to caspase-independent mechanisms for paclitaxel. ( A ) Inhibition of proteasome by bortezomib causes caspase-dependent apoptosis of macrophages followed by the release of HMGB1, which in turn causes neuronal sensitization via activation of RAGE and acceleration of CXCL12/CXCR4 signals, leading to CIPN. ( B ) Paclitaxel causes HMGB1 release from macrophages through activation of the ROS/p38MAPK/NF-κB pathway , independently of caspase (see D), and the extracellular HMGB1 develops CIPN in a manner dependent on RAGE and CXCR4 , as shown in the CIPN caused by bortezomib.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques: Inhibition, Activation Assay

Journal: Physiological Reports

Article Title: Tuberous sclerosis complex exhibits a new renal cystogenic mechanism

doi: 10.14814/phy2.13983

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Rabbit polyclonal Anti‐NCC , 1:60 , Stressmarq Biosciences; Victoria, BC, Canada , SPC‐402.

Techniques: Plasmid Preparation, Generated