Journal: Cell Reports

Article Title: The protease SPRTN and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability

doi: 10.1016/j.celrep.2021.110080

Figure Lengend Snippet:

Article Snippet: Rabbit anti-p97 , Proteintech , Cat#10736-1-AP; RRID: AB_2214635.

Techniques: Virus, Subcloning, Recombinant, Staining, Picogreen Assay, Proliferation Assay, Flow Cytometry, DNA Extraction, Sequencing, Luciferase, Software, Transfection, Modification, Magnetic Beads, Membrane

Journal: Brain : a journal of neurology

Article Title: Strumpellin is a novel valosin-containing protein binding partner linking hereditary spastic paraplegia to protein aggregation diseases.

doi: 10.1093/brain/awq222

Figure Lengend Snippet: Figure 2 (A) Strumpellin (stru) immunoblot analysis of a total protein extract from murine skeletal muscle (left) and mRFPmars immunodetection of mCherry-strumpellin overexpression in 293TN cells (right). (B) Immunoblot analysis of a strumpellin co-immunoprecipitation experiment. CoIP = co-precipitated proteins by the strumpellin antibody; ctrl = beads incubated with bovine serum albumin instead of polyclonal strumpellin antibody; input = soluble lysate of mCherry-strumpellin transfected 293TN cells; RFP = red fluorescent protein; stru = strumpellin; (1) = unspecific signal from the strumpellin antibody; (2) = degradation product of mCherry–strumpellin; (3) = signal from the strumpellin antibody heavy chain. (C) Pull-down experiment using recombinant GST–strumpellin coupled to beads and soluble VCP (GST-stru versus VCP). This experiment confirms a direct interaction between both proteins. As a negative control, beads were coated with GST (GST versus VCP). For illustration purposes individual lines from the original western blot were digitally re-arranged. (1) = strumpellin degradation products.

Article Snippet: Then sections were pre-incubated in 5% bovine serum albumin (0.3% Triton, 0.1 M phosphate-buffered saline, 45 min, room temperature) and incubated either with rabbit polyclonal anti-VCP antibody (dilution 1:500– 1:2000; NB100-1558, Novus Biologicals), rabbit polyclonal anti-strumpellin (dilution 1:50) or rabbit monoclonal anti-huntingtin antibody (1:500; clone EP867Y, Epitomics) diluted in 2% bovine serum albumin, 0.3% Triton, 0.1 M phosphate-buffered saline (48 h, 4 C).

Techniques: Western Blot, Immunodetection, Over Expression, Immunoprecipitation, Incubation, Transfection, Recombinant, Negative Control

Journal: Brain : a journal of neurology

Article Title: Strumpellin is a novel valosin-containing protein binding partner linking hereditary spastic paraplegia to protein aggregation diseases.

doi: 10.1093/brain/awq222

Figure Lengend Snippet: Figure 3 (A) Schematic structure of the human strumpellin gene. The human strumpellin (KIAA0196) locus comprises 68 kbp at position 126.1 Mb in chromosome 8q24.13, adjacent to the SQLE (squalene epoxidase) and NSMCE2 (non-SMC element 2) genes and linked within 1.5 Mbp to annexin A13 (NCBI reference NC_000008.19). It consists of one non-coding plus 28 protein-coding exons and an elevated incidence of phase 2 introns between codon base positions 2 and 3 in all vertebrate species examined. Known non-synonymous polymorphisms (nsSNPs) are indicated as P216 L, L229 R, N471D*, L619 F*, V626 F* (50 exon 16, 141 bp), P730 H and A1049 V identified by HapMap or the 1000 human genomes projects (*based on clinical data). (B) Schematic structure of the human strumpellin protein. Strumpellin consists of an N-terminal domain, a ‘spectrin-like’ repeat domain (five repeats), and a C-terminal domain. No structural similarity to known domains was found for the N-terminal domain. The strumpellin ‘spectrin-like’ repeat is predicted to be structurally similar to Exo70 (protein data bank accession number 2pft) and importin beta-2 (protein data bank accession number 2z5 k). The structure of Exo70 is shown for illustration only; the position of the polyclonal strumpellin antibody epitope (red) and the mutations known to cause hereditary spastic paraplegia (cyan) are highlighted at their approximate positions. Further known non-synonymous polymorphisms are indicated (black). The C-terminal domain of strumpellin has predicted similarity to exportin-5 (protein data bank accession number 3a6p) and importin beta-1 (protein data bank accession number 2 bpt). The structure of exportin-5 is also shown for illustration only. A potential splicing variant is indicated (M149). Prediction of secondary-structure elements of strumpellin as well as structural similarity searches were carried out using PSIPRED (Bryson et al., 2005). Structure-based sequence alignments for the spectrin repeats were generated manually using the structure of the 16th repeat from chicken brain alpha-spectrin [protein data bank accession number 1aj3; (Pascual et al., 1997)] as template. Figure prepared with PyMOL (DeLano, 2002).

Article Snippet: Then sections were pre-incubated in 5% bovine serum albumin (0.3% Triton, 0.1 M phosphate-buffered saline, 45 min, room temperature) and incubated either with rabbit polyclonal anti-VCP antibody (dilution 1:500– 1:2000; NB100-1558, Novus Biologicals), rabbit polyclonal anti-strumpellin (dilution 1:50) or rabbit monoclonal anti-huntingtin antibody (1:500; clone EP867Y, Epitomics) diluted in 2% bovine serum albumin, 0.3% Triton, 0.1 M phosphate-buffered saline (48 h, 4 C).

Techniques: Variant Assay, Sequencing, Generated

Journal: Brain : a journal of neurology

Article Title: Strumpellin is a novel valosin-containing protein binding partner linking hereditary spastic paraplegia to protein aggregation diseases.

doi: 10.1093/brain/awq222

Figure Lengend Snippet: Figure 9 Immunofluorescence analysis of IBMPFD muscle tissue (R155 C VCP mutation) and myofibrillar myopathies due to heterozygous desmin (R350 P), myotilin (S55 F) and B-crystallin (G154 S) mutations. Note that cytoplasmic strumpellin positive pathological protein aggregates (arrows) were present in all cases analysed. In IBMPFD strumpellin labelling was also observed in a VCP positive nucleus (double-arrowhead). Double-arrows denote subsarcolemmal strumpellin and desmin positive structures, whereas arrowheads demonstrate protein aggregates that are exclusively labelled by the strumpellin antibody. mAb = monoclonal antibody; pAb = polyclonal antibody.

Article Snippet: Then sections were pre-incubated in 5% bovine serum albumin (0.3% Triton, 0.1 M phosphate-buffered saline, 45 min, room temperature) and incubated either with rabbit polyclonal anti-VCP antibody (dilution 1:500– 1:2000; NB100-1558, Novus Biologicals), rabbit polyclonal anti-strumpellin (dilution 1:50) or rabbit monoclonal anti-huntingtin antibody (1:500; clone EP867Y, Epitomics) diluted in 2% bovine serum albumin, 0.3% Triton, 0.1 M phosphate-buffered saline (48 h, 4 C).

Techniques: Mutagenesis

Journal: Molecular Cancer Therapeutics

Article Title: Sorafenib-Mediated Targeting of the AAA+ ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

doi: 10.1158/1535-7163.mct-12-0516

Figure Lengend Snippet: Figure 5. Tyrosine phosphorylation of p97/VCP is negatively regulated by sorafenib. A, HepG2 cells were either untreated (CTL) or treated with 10 mmol/L sorafenib, 5 mmol/L BpVphen, or 10 mmol/L sorafenib combined with 5 mmol/L BpVphen for 4 hours. p97/VCP was immunoprecipitated from total cell lysates and immunoblots against phospho-tyrosine (pY) and p97/VCP were conducted. The amount of pY in each sample is shown at the bottom of the immunoblots as a percentage of CTL. A representative experiment out of 4 is shown (left). Quantification of the 4 experiments by densitometry represented as the mean SD. Statistical significance is shown: , P < 0.03; , P < 0.01 (right). B, cells were treated as earlier and the presence of p97/VCP in the total membrane fraction was analyzed by immunoblotting. Calnexin is shown as a loading control. A representative experiment out of 3 is shown (left). Quantification of the 4 experiments by densitometry represented as the mean SD. Statistical significance is shown: , P < 0.04; , P < 0.01 (right). C, same as in A, but BpVphen was replaced by 20 mmol/L DBeQ. D, HuH7 cells were either not treated (1) or treated with 1 mmol/L (4) or 10 mmol/L (7) sorafenib for 2 hours, 5 mmol/L BpVphen (2), 20 mmol/L DBeQ (3) for 3 hours or the combination of 1 mmol/L sorafenib þ 5 mmol/L BpVphen (5), 10 mmol/L sorafenib þ 5 mmol/L BpVphen (8), and 1 mmol/L sorafenib þ 20 mmol/L DBeQ (6). Cells were fixed and stained for Giantin (green) and Hoechst 33342 (blue). Images were acquired by confocal microscopy. Scale bars correspond to 10 mm.

Article Snippet: Rabbit polyclonal anti-Giantin and mouse monoclonal anti-p97 antibodies were purchased from Abcam.

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Membrane, Control, Staining, Confocal Microscopy

Journal: Molecular Cancer Therapeutics

Article Title: Sorafenib-Mediated Targeting of the AAA+ ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

doi: 10.1158/1535-7163.mct-12-0516

Figure Lengend Snippet: Figure 6. Sorafenib-mediated cell toxicity occurs in part through p97/VCP. A, HepG2 (left) and HuH7 (right) cells were incubated for 48 hours with increasing concentrations of sorafenib or DBeQ or a combination of an increasing concentration of sorafenib and 20 mmol/L DBeQ, and the cellular viability was analyzed using sulforhodamine B staining. B, HepG2 (left) and HuH7 (right) cells were treated with 2 mmol/L sorafenib and 20 mmol/L DBeQ for 36 hours. Apoptosis was determined using Annexin V staining and is represented as mean SD representative of 3 independent experiments. Statistical significance is shown: , P < 0.05; , P < 0.01. C, HuH7 cells were treated with 2 mmol/L sorafenib and 20 mmol/L DBeQ or the combination of both for 16 hours. LC3 maturation was assessed using immunoblot with anti-LC3 antibodies. A blot representative of 3 independent experiments is shown. D, schematic representation of sorafenib-mediated regulation of cell death through p97/VCP and the secretory pathway. OD, optical density.

Article Snippet: Rabbit polyclonal anti-Giantin and mouse monoclonal anti-p97 antibodies were purchased from Abcam.

Techniques: Incubation, Concentration Assay, Staining, Western Blot