Journal: Journal of Biological Chemistry

Article Title: Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle

doi: 10.1074/jbc.ra118.007287

Figure Lengend Snippet: Figure 2. Gene ontology (GO) term analysis of protein-protein interactions of wildtype and expanded PABPN1. A) Venn Diagram showing the number of proteins that interact with wildtype (Ala10) PABPN1 and alanine-expanded (Ala17) PABPN1. The number of proteins that interact to the same extent with Ala 10 and Ala17 PABPN1 (Ala10≈Ala17); more with Ala10 than Ala17 PABPN1 (Ala10>Ala17) and more with Ala17 than Ala10 PABPN1 (Ala17>Ala10) are indicated. B-D) The Gene Ontology (GO) terms for the proteins that are equally enriched (B) with wildtype (Ala 10) PABPN1 and alanine-expanded (Ala 17) PABPN1 (Ala10≈Ala17) as compared to an IgG control or preferentially enriched with (C) Ala10 (Ala10>Ala17) or (D) Ala17 (Ala17>Ala10) PABPN1 are shown. Inclusion criteria for this analysis are Z- score ≥ 2.0, p value < 0.00001, and ≥ 5 genes per GO term.

Article Snippet: Primary antibodies used in this study: Flag-M2 (1:1000, Sigma, F1804), HSP90 (1:4000, Santa Cruz, sc13119), Alanine (1:3000) (36), PABPN1 (1:3000) (22), hnRNPUL2 (1:1000, Bethyl, A304-619A), PABPC1 (1:2000, Abcam, ab21060), TDP-43 (1:2000, Proteintech, 10782- 2-AP) and Progranulin (1:2000, Novus Biologicals, 26320002) Liquid chromatography and tandem mass spectrometry We performed on-bead mass spectrometry to identify PABPN1 interacting proteins.

Techniques: Protein-Protein interactions, Control

Journal: Journal of Biological Chemistry

Article Title: Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle

doi: 10.1074/jbc.ra118.007287

Figure Lengend Snippet: Figure 4. Protein interactions of wildtype (Ala10) PABPN1 and alanine-expanded (Ala17) PABPN1. A) To examine interaction with endogenous PABPN1, skeletal muscle (Gastrocnemius) lysate was incubated with control IgG or an antibody against PABPN1 (22). Input and bound fractions were resolved by SDS-PAGE and immunoblotted with anti-PABPN1, anti-hnRNPUL2 or anti-SKIP/SNW antibody. Shown is a representative image from n = 2 immunoblots using lysate from four electroporated mice. B) To examine differential interactions with wildtype and alanine-expanded PABPN1, lysates obtained from muscles electroporated with plasmids expressing wildtype (Ala10) or alanine-expanded (Ala17) PABPN1 were incubated with anti-Flag antibody conjugated beads. Bound fractions were resolved by SDS-PAGE followed by immunoblotting with anti-Flag, anti-PABPC1 and anti-TDP-43 antibodies. Shown is a representative image from n = 2 immunoblots using lysate from four electroporated mice. C) Lysates obtained from muscles electroporated with plasmids expressing Flag-tagged wildtype (Ala10) or alanine- expanded (Ala17) PABPN1 were analyzed by sedimentation through 10-30% glycerol gradients. Alternate gradient fractions from top to bottom were loaded from left to right together with an input lane and resolved by SDS-PAGE. Immunoblotting with an anti-Flag antibody was used to detect wildtype (Ala 10) PABPN1 or alanine-expanded (Ala 17) PABPN1 in the fractions. Shown is a representative immunoblot from three separate fractionation experiments. The box highlights Ala17 PABPN1 present in high molecular weight complexes.

Article Snippet: Primary antibodies used in this study: Flag-M2 (1:1000, Sigma, F1804), HSP90 (1:4000, Santa Cruz, sc13119), Alanine (1:3000) (36), PABPN1 (1:3000) (22), hnRNPUL2 (1:1000, Bethyl, A304-619A), PABPC1 (1:2000, Abcam, ab21060), TDP-43 (1:2000, Proteintech, 10782- 2-AP) and Progranulin (1:2000, Novus Biologicals, 26320002) Liquid chromatography and tandem mass spectrometry We performed on-bead mass spectrometry to identify PABPN1 interacting proteins.

Techniques: Incubation, Control, SDS Page, Western Blot, Muscles, Expressing, Sedimentation, Fractionation, High Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle

doi: 10.1074/jbc.ra118.007287

Figure Lengend Snippet: Figure 5. TDP-43 protein physiology is altered in myoblasts expressing Ala17 PABPN1. A) Primary myoblasts isolated from wildtype (Ala10/Ala10) or OPMD model (Ala17/Ala10) mice were differentiated into myotubes and lysed in a gentle lysis buffer. The Soluble (S) and Pellet (P) fractions following centrifugation were resolved by SDS-PAGE and immunoblotted to detect TDP-43. Top and bottom bands represent TDP-43 full length and 35 kDa cleavage product, respectively (90). Shown is a representative immunoblot for three independent experiments. B) Soluble lysates obtained from wildtype (Ala10/Ala10) or OPMD (Ala17/Ala10) myotubes prepared in (A) were resolved on density glycerol gradients and fractions were collected from top to bottom. Input samples and alternate fractions were resolved by SDS- PAGE and immunoblotted for TDP-43. Shown is a representative immunoblot for three independent experiments. C) Myoblasts isolated from wildtype (Ala10/Ala10) and OPMD (Ala17/Ala10) mice were cultured in the absence (-) or presence (+) of 1 mM hydrogen peroxide followed by lysis in a non-reducing buffer (-DTT). Lysates were resolved by SDS-PAGE and immunoblotted for TDP-43 to detect low molecular weight (LMW) and high molecular weight (HMW) species of TDP-43. Shown is a representative immunoblot for three independent experiments. D) Limb muscles from wildtype and Transgenic Ala 17.1 mice (34) were sectioned and permeabilized. Sections were left untreated (-KCl) or treated with 1 M KCl in HPEM buffer (+KCl) to remove soluble proteins, followed by detection of TDP-43 with anti-TDP-43 antibody. KCl-insoluble aggregates are indicated by white arrowheads and are magnified in the inset. DAPI and dystrophin staining were used to mark the nucleus and sarcolemma, respectively.

Article Snippet: Primary antibodies used in this study: Flag-M2 (1:1000, Sigma, F1804), HSP90 (1:4000, Santa Cruz, sc13119), Alanine (1:3000) (36), PABPN1 (1:3000) (22), hnRNPUL2 (1:1000, Bethyl, A304-619A), PABPC1 (1:2000, Abcam, ab21060), TDP-43 (1:2000, Proteintech, 10782- 2-AP) and Progranulin (1:2000, Novus Biologicals, 26320002) Liquid chromatography and tandem mass spectrometry We performed on-bead mass spectrometry to identify PABPN1 interacting proteins.

Techniques: Expressing, Isolation, Gentle, Lysis, Centrifugation, SDS Page, Western Blot, Cell Culture, Molecular Weight, High Molecular Weight, Muscles, Transgenic Assay, Staining

Journal: Journal of Biological Chemistry

Article Title: Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle

doi: 10.1074/jbc.ra118.007287

Figure Lengend Snippet: Figure 6. TDP-43 regulated alternative splicing is altered in cells expressing alanine-expanded PABPN1. A) Analysis of RT-PCR products of alternatively spliced exons of Mef2D (exon 8), Sort1 (exon 17b) and Dnajc5 (exon 4) using RNA isolated from wildtype Ala10/Ala10 (A10/A10) or OPMD Ala17/Ala10 (A17/A10) primary mouse myoblasts. Gapdh serves as a loading control. The upper and lower bands are the exon-included (+) and exon-excluded (-) PCR products, respectively. B) Densitometric quantification of the exon-included and exon-excluded RT-PCR products from (A) is presented as a ratio of Included/Excluded PCR products. Shown is mean plus or minus standard deviation for n = 3; * indicates p<0.05. C) Quantitative RT- PCR analysis of Sort1 alternative splicing in wildtype (A10/A10) or OPMD (A17/A10) primary mouse myoblasts. The steady state level of Sort1 RNA with exon 17b included or excluded was quantified by qRT-PCR using variant-specific primers and is represented as the Sort1 Included/Excluded ratio normalized to Gapdh. Data shown represent mean plus or minus standard deviation for n = 16. * indicates p<0.05.

Article Snippet: Primary antibodies used in this study: Flag-M2 (1:1000, Sigma, F1804), HSP90 (1:4000, Santa Cruz, sc13119), Alanine (1:3000) (36), PABPN1 (1:3000) (22), hnRNPUL2 (1:1000, Bethyl, A304-619A), PABPC1 (1:2000, Abcam, ab21060), TDP-43 (1:2000, Proteintech, 10782- 2-AP) and Progranulin (1:2000, Novus Biologicals, 26320002) Liquid chromatography and tandem mass spectrometry We performed on-bead mass spectrometry to identify PABPN1 interacting proteins.

Techniques: Alternative Splicing, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Control, Standard Deviation, Quantitative RT-PCR, Variant Assay

Journal: Journal of Biological Chemistry

Article Title: Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle

doi: 10.1074/jbc.ra118.007287

Figure Lengend Snippet: Figure 7. SORT1 protein function is altered in muscle cells that express alanine-expanded PABPN1. A) Wildtype (Ala10/Ala10) and OPMD (Ala17/Ala10) myoblasts and myotubes were cultured in the presence of a fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) for 6 hours. Cellular fluorescence (RFU) was measured with a plate reader as a reporter for glucose uptake. Shown is mean plus or minus standard deviation for n = 3 experiments. * indicates p<0.05. B) Wildtype (Ala10/Ala10) and OPMD (Ala17/Ala10) myotubes were cultured in the presence or absence of recombinant N-TAP human progranulin (PGRN) for 16 hours. Cell lysates were resolved by SDS-PAGE followed by immunoblotting for PGRN for three independent samples for each condition analyzed. C) Band intensities for PGRN uptake from (B) were quantified and normalized to total protein (Ponceau S staining). Shown is mean plus or minus standard deviation for n = 4 experiments. * indicates p<0.05. D) To control for the specificity of PGRN uptake, wildtype (Ala10/Ala10) and OPMD (Ala17/Ala10) myotubes were cultured in the presence or absence of recombinant N-terminally TAP-tagged (N-TAP) or C-terminally- TAP-tagged (C-TAP) human progranulin (PGRN) for 16 hours. Cell lysates were resolved by SDS-PAGE followed by immunoblotting for PGRN. HSP90 serves as a loading control.

Article Snippet: Primary antibodies used in this study: Flag-M2 (1:1000, Sigma, F1804), HSP90 (1:4000, Santa Cruz, sc13119), Alanine (1:3000) (36), PABPN1 (1:3000) (22), hnRNPUL2 (1:1000, Bethyl, A304-619A), PABPC1 (1:2000, Abcam, ab21060), TDP-43 (1:2000, Proteintech, 10782- 2-AP) and Progranulin (1:2000, Novus Biologicals, 26320002) Liquid chromatography and tandem mass spectrometry We performed on-bead mass spectrometry to identify PABPN1 interacting proteins.

Techniques: Cell Culture, Fluorescence, Standard Deviation, Recombinant, SDS Page, Western Blot, Staining, Control