Journal: European journal of neurology

Article Title: Expression of vesicle-associated membrane-protein-associated protein B cleavage products in peripheral blood leukocytes and cerebrospinal fluid of patients with sporadic amyotrophic lateral sclerosis.

doi: 10.1111/ene.12334

Figure Lengend Snippet: Figure 1 Schematic representation of structural domains of human VAPB protein. Positions of VAPB mutations (p.P56S, p.T46I and p.V234I) and immunogen peptide (31–180 aa) of the wild-type VAPB used to generate the anti-human VAPB antibody (Proteintech Group Inc.) are shown.

Article Snippet: Membranes blocked with TBS buffer plus 0.1% Tween-20 and 5% non-fat milk for 1 h were incubated with a polyclonal rabbit anti-human (Nterminal) VAPB antibody (1:400, Proteintech Group Inc., Chicago, IL, USA) overnight at 4°C.

Techniques:

Journal: European journal of neurology

Article Title: Expression of vesicle-associated membrane-protein-associated protein B cleavage products in peripheral blood leukocytes and cerebrospinal fluid of patients with sporadic amyotrophic lateral sclerosis.

doi: 10.1111/ene.12334

Figure Lengend Snippet: Figure 2 Expression of VAPB and VAPA proteins in PBL and CSF from healthy (HC) and neurological (NC) controls. (a), (b), (d) Representative immunoblots showing the expression pattern of VAPB (a), (b) and VAPA (d) identified in PBL and CSF. (c) Peptide competition experiments to verify specificity of the anti- VAPB antibody. Western blots were carried out with (a) 100 lg or (b)–(d) 65 lg proteins of extracts. Differences in the molecu- lar sizes of VAPB and VAPA bands are reported in leucocytes. VAPB and VAPA exhibit two close bands that approximate the molecular size of 17 kDa. In CSF, only the 14 kDa band of VAPB was identified in most samples examined, and no VAPA product was detected; in rare instances, a 24 kDa band of VAPB (asterisk) was additionally detected.

Article Snippet: Membranes blocked with TBS buffer plus 0.1% Tween-20 and 5% non-fat milk for 1 h were incubated with a polyclonal rabbit anti-human (Nterminal) VAPB antibody (1:400, Proteintech Group Inc., Chicago, IL, USA) overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: European journal of neurology

Article Title: Expression of vesicle-associated membrane-protein-associated protein B cleavage products in peripheral blood leukocytes and cerebrospinal fluid of patients with sporadic amyotrophic lateral sclerosis.

doi: 10.1111/ene.12334

Figure Lengend Snippet: Figure 3 Scatter plots of the expression levels of VAPB MSP fragments in PBL of SALS patients and controls. Levels of the 17 kDa (a) or 14 kDa (b) fragments are expressed as the ratio between their intensities and those of b-actin immunoreactive bands. No significant differences for the two fragments were found amongst HC, NC and SALS.

Article Snippet: Membranes blocked with TBS buffer plus 0.1% Tween-20 and 5% non-fat milk for 1 h were incubated with a polyclonal rabbit anti-human (Nterminal) VAPB antibody (1:400, Proteintech Group Inc., Chicago, IL, USA) overnight at 4°C.

Techniques: Expressing

Journal: European journal of neurology

Article Title: Expression of vesicle-associated membrane-protein-associated protein B cleavage products in peripheral blood leukocytes and cerebrospinal fluid of patients with sporadic amyotrophic lateral sclerosis.

doi: 10.1111/ene.12334

Figure Lengend Snippet: Figure 4 Analyses of the VAPB MSP fragment in CSF from neurological controls (NC) and SALS patients with bulbar (ALS-B) and spinal onset (ALS-S). (a) Representative immunoblots of the CSF 14 kDa fragment from 15 subjects analysed of each group; albumin immunoblotting was used to control protein loading. (b), (c) Scatter plots of the expression levels of the CSF VAPB MSP fragment detected in the samples examined (NC, n = 33; SALS, n = 46; ALS-S, n = 22; ALS-B, n = 24). Western blot analyses of VAPB MSP were performed in triplicate using the same amount of proteins (65 lg) for each CSF sample and by reprobing membranes with albu- min antibody. Levels of the 14 kDa fragment result from the ratio of the fragment over albumin density in each sample on western blotting. (a) *Mann–Withney U test (P < 0.0001, NC vs. SALS); (b) *Kruskal–Wallis test with Dunn’s multiple comparison test (P < 0.0001, NC vs. ALS-B).

Article Snippet: Membranes blocked with TBS buffer plus 0.1% Tween-20 and 5% non-fat milk for 1 h were incubated with a polyclonal rabbit anti-human (Nterminal) VAPB antibody (1:400, Proteintech Group Inc., Chicago, IL, USA) overnight at 4°C.

Techniques: Western Blot, Control, Expressing, Comparison

Journal: PloS one

Article Title: The carboxyl-terminal amino acids render pro-human LC3B migration similar to lipidated LC3B in SDS-PAGE.

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Figure 1. Schematic diagram of expression plasmid constructs. Myc, GFP, or 3xFlag tag is fused to human MAP1LC3B ORF at its N- terminus. The box bars on the left represent the N-terminal tags. The dashed lines represent MAP1LC3B with 27 amino acids sequence shown at the carboxyl terminus. The arrow points to Glycine 120 conjugation site. The last five amino acids at the C-terminal are underlined. Mutant amino acid or last amino acid of LC3B is bolded. Human LC3C, Rat LC3, Rat LC3B, Mouse LC3A, Mouse LC3B and the related mutants are described in Result section. doi:10.1371/journal.pone.0074222.g001

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from Nterminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e74222 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and b-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Expressing, Plasmid Preparation, Construct, Sequencing, Conjugation Assay, Mutagenesis

Journal: PloS one

Article Title: The carboxyl-terminal amino acids render pro-human LC3B migration similar to lipidated LC3B in SDS-PAGE.

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Figure 2. G120A mutant of human LC3B has similar mobility to LC3B-II in SDS-polyacrylamide gel. Aa, plasmids expressing Myc-LC3B, myc-LC3BG120A or Myc-LC3BG120 was expressed in HEK293 cells for 24 hours. Cells were harvested and lysed by sonication, and 10 mg of total protein of each lysate were separated by 15% of SDS-polyacrylamide gel, followed by immunoblotting with anti-LC3B antibody. Ab, plasmids expressing Flag-LC3B or Flag-LC3BG120A was expressed in HEK293 cells for 24 hours. Cells were lysed followed by immnuoblotting using Flag-antibody. B, plasmids expressing LC3B, LC3BG120A or LC3BA120 was expressed in HEK293 cells for 24 hours. Cells were treated with 50 mM of CQ for 2 hours before collection. Transient expressed proteins were analyzed by immunoblotting using LC3B antibody.Ca, plasmids expressing GFP-LC3B or GFP-LC3G120A

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from Nterminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e74222 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and b-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Mutagenesis, Expressing, Sonication, Western Blot

Journal: PloS one

Article Title: The carboxyl-terminal amino acids render pro-human LC3B migration similar to lipidated LC3B in SDS-PAGE.

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Figure 3. Analysis of different group amino acid replacement of Glycine 120 of human LC3B and LC3B truncates effect on their mobility in SDS-polyacrylamide gel. A, plasmids expressing Flag-LC3B, Flag-LC3BG120A, Flag-LC3BG120 or Flag-LC3A120 was expressed in HEK293 cells for 24 hours. Cells were treated with 50 mM of CQ for two hours and harvested and lysed. Total cell lysates (10 mg) was used for immunoblotting by Flag antibody. B, G120 point mutant plasmids were expressed in HEK293 cells for 24 hours. Immunoblotting was conducted to compare the band’s mobility with Flag-LC3B, Flag-LC3BG120A or Flag-LC3BG120A as indicated. C, Flag-LC3BG120D and Flag-LC3BG120E were expressed in HEK293 cells for 24 hours. Cells were then treated with 50 mM of CQ for 2 hours. Expression patters of Flag-LC3BG120D and Flag-LC3BG120E were compared with endogenous LC3B by immunoblotting with LC3B antibody (upper panels). Adenoviral vector expressing GFP-LC3B, GFP-LC3BG120D or GFP-LC3BG120E

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from Nterminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e74222 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and b-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Expressing, Western Blot, Mutagenesis, Plasmid Preparation

Journal: PloS one

Article Title: The carboxyl-terminal amino acids render pro-human LC3B migration similar to lipidated LC3B in SDS-PAGE.

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Figure 5. Pro-human LC3B migrates at similar rate as G120A mutant and lipidated form in SDS-polyacrylamide gel. A, pTriEX.1.1 plasmids encoding Flag-LC3B, Flag-LC3BG120A, or Flag- LC3BA120 was expressed in HEK293 or E.coli cells (Tuner cell), respectively. Recombinant protein expression in E.coli cells were induced with 1 mM of IPTG for four hours before harvesting. 10 mg of HEK293 or E.coli lysates were loaded onto SDS-polyacrylamide gel. Immunoblotting was conducted using Flag-antibody. B, 10 mg of E.coli lysates with Flag-LC3B or Flag-LC3BG120A recombinant proteins was digested with recombinant Atg4B at 37uC for indicated time and analyzed by immunoblotting with LC3B antibody and Atg4B antibody as indicated. doi:10.1371/journal.pone.0074222.g005

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from Nterminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e74222 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and b-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Mutagenesis, Recombinant, Expressing, Western Blot

Journal: PloS one

Article Title: The carboxyl-terminal amino acids render pro-human LC3B migration similar to lipidated LC3B in SDS-PAGE.

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Figure 7. Sequence comparison of LC3 family member from human, mouse and rat origins. Pairwise alignments of LC3A, LC3B and LC3C of human and rodent origin are generated by ClustalW2. The glycine conjugation sites are boxed and C-terminal amino acids after glycine conjugation site are underlined. A ‘‘*’’ presents identity of amino acids; ‘‘:’’ presents these amino acids in a same amino acid group (similarity); ‘‘?’’ presents different group of amino acids. doi:10.1371/journal.pone.0074222.g007

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from Nterminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e74222 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and b-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Sequencing, Comparison, Generated, Conjugation Assay

Journal: PloS one

Article Title: The carboxyl-terminal amino acids render pro-human LC3B migration similar to lipidated LC3B in SDS-PAGE.

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Figure 8. The faster migration is conserved in pro-LC3B of rodent species but not in human LC3C. A, Compare pro-human LC3B with pro-mouse LC3B, pro-rat LC3B and their cleaved and lipidated forms’ band patterns by 15% of SDS-polyacrylamide gel. Indicated expression plasmids were expressed in HEK293 cells. B, compare pro-rat LC3B with pro-rat LC3 and cleaved forms by 15% of SDS-polyacrylamide gel. Correspondent expression plasmids were expressed in HEK293 cells. Note: human LC3B-I band is close to RatLC3B-I/RatLC3-I as indicated by arrows. C, pro-LC3A and its cleaved form comparison by 15% of SDS-polyacrylamide gel. Indicated expression plasmids were expressed in HEK293 cells. D, compare pro-

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from Nterminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e74222 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and b-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Migration, Expressing, Comparison

Journal: PloS one

Article Title: The carboxyl-terminal amino acids render pro-human LC3B migration similar to lipidated LC3B in SDS-PAGE.

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Figure 9. Effect of Atg4B on LC3B processing. A, MEFwt and MEFatg7KO cells were treated with 50 mM of CQ for 2 hours. LC3B expression patterns were then analyzed in 15% of SDS-PAGE by immunoblotting with LC3B antibody. B, Adenoviral vector expressing GFP-LC3B was used to infect MEFatg7KO cells in series double diluted doses for 24 hours (D1 = 20MOI). Ad-GFP-LC3B (20MOI) infected wild-type MEF cells were used as control or treated with 50 mM of CQ for 2 hours.GFP-LC3B expression patterns were analyzed by 15% of SDS-PAGE. C, Ad-GFP-LC3B infected MEFwt and MEFatg7KO cells were treated with 50 mM of CQ for 2 hours and images were recorded by fluorescence microscopy. D, HEK293 cells were transfected with Atg4B siRNA and Control siRNA for 48 h. 15 mg of total cell lysates were analyzed by immunoblotting with Atg4B, LC3B, and b-actin antibodies. Image J software was used to quantify band density using b-actin as loading control. E, Diagram of LC3 species migration patterns in 15% of SDS-polyacrylamide gel. The diagram summarizes LC3 species (pro-LC3, LC3-I and LC3-II) band patterns from human, mouse and rat origins. Refer to the result section for description in details. doi:10.1371/journal.pone.0074222.g009

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from Nterminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e74222 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and b-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Expressing, SDS Page, Western Blot, Plasmid Preparation, Infection, Control, Fluorescence, Microscopy, Transfection, Software, Migration