flag-sam68 vector  (Thermo Fisher)

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: SAM68 binds SMN2 exon 7 and mediates recruitment of hnRNP A1 in vivo. (A) Schematic representation of the human SMN2 gene. Boxes represent exons, black lines represent introns, and the red box indicates the regulated exon 7. Red arrows indicate the oligonucleotide pairs used in the analysis. (B) CLIP assay of SAM68-bound SMN2 pre-mRNA in brain of non-SMA mice ( SMN2Δ7;SMN2;Smn +/+ ). (C) CLIP assay of hnRNP A1 in brain of non-SMA mice that are wild type or knockout for Sam68 . Signals for SAM68 (B) and hnRNP A1 (C) binding was calculated as fold enrichment versus IgGs and expressed as mean ± SD; n = 3. The p-value was determined by two-tailed t test (B) or one-way ANOVA test followed by Bonferroni’s multiple comparison posttest (C). *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant (P > 0.05). (D) Sequence of human SMN2 probe used to synthesize the biotinylated RNA for the streptavidin pull-down experiment. Lowercase letters indicate the intron 6 sequence; uppercase letters indicate the exon 7 sequence. Bold letters and red arrows indicate primers used to synthesize the probe, and red bold letters indicate the exonic splicing silencers created by the C to T transition in SMN2 to which SAM68 binds. (E and F) Western blot analysis of the binding of endogenous U2AF65 to the biotinylated probe (SMN2 Ex7) in streptavidin pull-down assays using brain extracts from non-SMA Sam68 +/+ (wt) or Sam68 −/− (ko) mice (E) or extracts from HEK293T cells transfected (+) or not (−) with FLAG-SAM68 (F). Results are representative of three experiments that yielded similar results. ko, knockout; N.E., nuclear extracts; wt, wild type.

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: In Vivo, Knock-Out, Binding Assay, Two Tailed Test, Sequencing, Western Blot, Transfection

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Ablation of Sam68 expression partially rescues viability, body weight, and motor function of SMAΔ7 mice. (A) Schematic diagram of transgenic mice crossing (top) and PCR analysis of genomic DNA (bottom) from tails of SMN2Δ7;SMN2;Smn +/+ ;Sam68 +/+ (first lane), SMN2Δ7;SMN2;Smn −/− ;Sam68 +/+ (second lane), and SMN2Δ7;SMN2;Smn −/− ;Sam68 −/− (third lane) mice. Bands showing the transgenic and endogenous DNA bands amplified are indicated; the asterisk indicates an undefined band present in SMAΔ7 transgenic mice. (B) qPCR of SMN2Δ7 to quantify the dosage of transgenic SMN2Δ7 in hemizygous and homozygous transgenic animals. The data shown are from a single representative experiment, and values were normalized with Apolipoprotein B mRNA. (C) Micrograph showing representative non-SMA and SMAΔ7 (SMA) mice that are either wild type ( Sam68 +/+ ) or knockout ( Sam68 −/− ) for Sam68 . (D) Kaplan-Meier survival curves of SMAΔ7/ Sam68 +/+ ( n = 50) and SMAΔ7/ Sam68 −/− ( n = 22). Statistical analysis was performed by the log-rank test (P < 0.0001). (E) Weight curves of non-SMA/ Sam68 +/+ , non-SMA/ Sam68 −/− , SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− mice. Plot shows mean ± SD of at least 15 mice for each group for each day analyzed. The p-values were determined by a two-way ANOVA test followed by Bonferroni’s multiple comparison posttest. # represents the p-value of the non-SMA/ Sam68 +/+ versus non-SMA/ Sam68 −/− comparison; § represents the p-value of the non-SMA/ Sam68 −/− versus SMAΔ7/ Sam68 −/− comparison; and * represents the p-value of the SMAΔ7/ Sam68 +/+ versus SMAΔ7/ Sam68 −/− comparison. For all comparisons, P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 are represented by increasing symbols (from one to four). (F) Time required for pups to stand up after being placed on their sides for SMAΔ7/ Sam68 +/+ mice compared with SMAΔ7/ Sam68 −/− mice at 8 and 10 dpp (nd [nondetected] indicates experimental animals that never stood up during the test). Statistical analysis was performed by two-way ANOVA test followed by Bonferroni’s multiple comparison posttest (*, P < 0.05; ***, P < 0.001).

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Transgenic Assay, Amplification, Knock-Out

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Sam68 deletion rescues SMN2 splicing and expression in SMAΔ7 tissues. (A) qPCR analysis of exon 7–containing SMN2 transgene transcripts normalized to constant exon 6 in tissues of 10-dpp SMAΔ7/ Sam68 +/+ and SMAΔ7/ Sam68 −/− mice. Bar graph represents mean ± SD. n = 3. The p-value was determined by two-tailed t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (B) Western blot analysis of SMN protein expression in the indicated tissues of 10-dpp SMAΔ7/ Sam68 +/+ (wt) or SMAΔ7/ Sam68 −/− (ko) mice. Actin was used as a loading control. Quantification of the SMN band is shown at the bottom. Each point value represents the mean ± SD. n = 2. Cereb, cerebellum; ko, knockout; S.cord, spinal cord; wt, wild type.

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Two Tailed Test, Western Blot, Knock-Out

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Ablation of Sam68 rescues SMN assembly into nuclear gems in spinal cord motor neurons. (A) Immunodetection of SAM68 (blue) in ChAT-positive motor neurons (red) in the lumbar spinal cord (L1–L5) of 8-dpp non-SMA mice. DAPI was used for staining of nuclei. Motor neurons are indicated by arrows and surrounding cells by arrowheads. (B) Immunodetection of SMN gems (green) in ChAT-positive motor neurons (red) in the spinal cord (L1–L5) of 8-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. DAPI was used for staining of nuclei. Nuclear gems are indicated by arrowheads. Bars: (A and B) 20 µm; (insets) 10 µm. (C) Quantitative analysis of SMN gems in spinal motor neurons. The bar graph (mean ± SD; n = 3) shows the number of gems per 100 nuclei analyzed per sample (top) and the percentage of gem-positive motor neurons (bottom). Statistical analysis was performed by one-way ANOVA test followed by Bonferroni’s multiple comparison posttest (*, P < 0.05; ***, P < 0.001).

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Immunodetection, Staining

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Ablation of Sam68 rescues motor neuron loss in the spinal cord of SMAΔ7 mice. (A) Schematic representation of a Nissl-stained spinal cord section of the lumbar spinal cord from 8-dpp non-SMA mice (right). The red circle highlights the ventral horn of the spinal cord analyzed. Bar, 250 µm. (B) Higher magnification of sections of the ventral lumbar spinal cord from 8-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. Motor neurons are indicated by arrowheads. Bar, 125 µm. (C) Bar graph representing motor neuron counts (mean ± SD; n = 3) in lumbar spinal cord from mice described in B. Statistical analysis was performed by one-way ANOVA test followed by Bonferroni’s multiple comparison posttest (***, P < 0.001; ****, P < 0.0001).

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Ablation of Sam68 ameliorates innervation of NMJs. (A) NMJ immunofluorescence images from whole-mount FDB2 of 10-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. Higher magnifications are shown in the bottom panels. Postsynaptic (AChRs stained with α-bungarotoxin; green), presynaptic (stained with α-synaptophysin; red), and motor neuron axons (stained with an antibody against the heavy chain of neurofilament; red) were visualized. Bars, 100 µm. Arrows point to innervated NMJs (yellow staining), and arrowheads point to denervated NMJs (green staining). (B) Bar graph showing the percentage of innervated (left) and denervated (right) NMJs in FDB2 of non-SMA, non-SMA/ Sam68 −/− , SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− mice (for all, n = 3). Quantification of NMJ innervation was performed on at least 100 optical sections for mice, with a step size of 3 µm from the whole FDB2 of each genotype. Data represent mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s multiple comparison posttest (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant [P > 0.05]).

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Immunofluorescence, Staining

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Skeletal muscle atrophy is ameliorated in the SMAΔ7/ Sam68 −/− mice. (A) Hematoxylin and eosin staining of triceps muscle sections from 10-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. Bar, 100 µm. (B) Frequency distribution of muscle fiber areas (at least 2,000 for each genotype) of the experimental animals described in A. The colored lines and dotted line (non-SMA mice) represent the median value for each genotype: non-SMA mice ( n = 3) median size = 531 µm 2 and fiber size (mean ± SEM) = 568.76 ± 5.8 µm 2 ; non-SMA/ Sam68 −/− mice ( n = 3) median size = 557 µm 2 and fiber size (mean ± SEM) = 586.60 ± 5.7 µm 2 ; SMAΔ7/ Sam68 +/+ median size = 327 µm 2 and fiber size (mean ± SEM) = 342.32 ± 3.1 µm 2 ; and SMAΔ7/ Sam68 −/− median size = 486 µm 2 and fiber size (mean ± SEM) = 503.59 ± 4.6 µm 2 . Statistical analysis of the median values among groups was performed by one-way ANOVA test followed by Dunn’s multiple comparison posttest. (C) qPCR analysis of the expression of atrophy-related genes in quadriceps and lower leg muscles of mice described in B. Values (mean ± SD; n = 4) were normalized with Actin mRNA. Statistical analysis was performed by one-way ANOVA test followed by Bonferroni’s multiple comparison posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant (P > 0.05). r.l., relative level.

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Staining, Expressing