![]() Expression profiles for 2573 DEGs with abs (logFC) > 0.5, detected in contrast untreated Gaa KO DBA vs. ![]() ![]() (b-g) Gaa WT DBA ( n = 4), Gaa KO DBA ( n = 5), Gaa KO DBA +AAV ( n = 4). Western blot quantification is shown in panels e (GAA), f (p62) and g (Parkin). The lanes were run on the same gel but were non-contiguous. The molecular weight marker (kDa) is depicted. (d) Western blot analyses of triceps lysates with anti-human GAA, anti-Parkin, and anti-p62 antibodies, anti-Gapdh antibody was used as loading control. (c) Analyses of glycogen storage in triceps muscle. (b) Analyses of GAA enzyme activity in triceps muscle. Biochemical (b-g) and downstream analysis of RNA sequencing (RNA-seq) data (h-i) of skeletal muscle (SkM) at the end of the study. Vectors were delivered at postnatal day 2 (P2), analyses were performed 4 months (4M) later SkM (skeletal muscle), SC (spinal cord). 4 (a) Study diagram: Gaa KO DBA mice were treated by intravenous injection of AAV9 vectors encoding for a codon-optimized secretable human GAA (secGAA) under the control of the tandem liver-muscle LiMP promoter (AAV-secGAA, dose: 4 x 10 10 vg/mouse, ~2 x 10 13 vg/kg) littermate untreated (no Tx) Gaa KO DBA and Gaa WT DBA were used as controls. 4 Gene therapy with AAV vectors encoding for a secretable GAA variant rapidly rescues skeletal muscle defects in Gaa KO DBA mice. iNs, induced neurons Tuj1, class III beta-tubulin MAP2, microtubule-associated protein 2 HFF, human foreskin fibroblast NeuN, neuronal nuclei GABA, gamma-aminobutyric acid vGLUT1, vesicular glutamate transporter 1 GFAP, glial fibrillary acidic protein DCX, doublecortin Ascl1, achaete-scute family bHLH transcription factor 1 CTGF, connective tissue growth factor DKK3, dickkopf WNT signaling pathway inhibitor 3 COL1a1, collagen type 1 alpha-1 chain.įig. Unpaired Student's t-tests were used to compare data. (E) Graphs show the semi-quantification of the western blot analysis. (D) Western blot analysis of the protein expression levels of Tuj1, NeuN and GAPDH in HFFs and iNs. (C) Reverse transcription-quantitative PCR analysis revealed that in comparison with HFFs, there was a significant increase in gene expression levels of MAP2, DCX, NeuroD1 and ASCL1 in iNs, whereas human fibroblast-specific genes were downregulated in iNs on day 30. (B) iNs were stained for NeuN, GABA, vGLUT1 and GFAP (all green) on day 30. (A) iNs exhibited bipolar neuronal morphologies and expressed tuj1 (red) and MAP2 (green) at different phases (day 7, 14 and 30). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).įigure 3. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). A 37 kDa band corresponding to GAPDH was observed across the cell lines and tissues tested. The blot was probed with Anti-GAPDH Polyclonal Antibody (Product # PA1-988, 1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Western blot analysis was performed on whole cell extracts (30 µg lysate) of A549 (Lane 1), COS-7 (Lane 2), MDCK (Lane 3), HT-29 (Lane 4), C2C12 (Lane 5), PC-12 (Lane 6), U-2 OS (Lane 7) and tissue extract (30 µg lysate) of Rat Testis (Lane 8), Mouse Liver (Lane 9).
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