Journal: Frontiers in Molecular Neuroscience

Article Title: Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: a study of DNAJB6 chaperone

doi: 10.3389/fnmol.2015.00040

Figure Lengend Snippet: Canonical members of HSPB family prevent the aggregation of Aβ-GFP. Cells were transfected with HSPB1, HSPB5, HSPB7 or FRTTO at 1:3 ratio for 48 h. Quantification of the pellet/soluble ratio of Aβ-GFP relative to FRTTO was depicted in the chart above the blot. Values represent mean ± SE of two independent experiments. ** P < 0.01, ns = non significant.

Article Snippet: The membranes were blocked with 5% dry milk in PBS with 0.1% Tween 20 (PBST) for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: 6E10 (1:1000 in TBST, Covance), anti V5 (1:5000 in PBST, Invitrogen), anti β-actin (1:1000 in PBST, Abcam), anti HSPB1 (1:1000 in PBST, Stress Marq Biosciences), anti HSPB5 (1:1000 in PBST, Stress Marq Biosciences) and anti HSPB7 (1:1000, Abnova).

Techniques: Transfection

Journal: bioRxiv

Article Title: Peripherally expressed misfolded proteins remotely disrupt brain function and aggravate stroke-induced brain injury

doi: 10.1101/785477

Figure Lengend Snippet: a Protein aggregates in primary neuronal cultures detected with a Proteostat aggresome detection kit following incubation with the exosomes isolated from CryAB R120G mice. b Quantitative analysis of a . c Cultured neurons treated with CryAB R120G exosomes showed a reduced ATP level. d Schematic illustration of an cell culture assay to determine the prion-like property of CryAB R120G protein. e Immunocytochemical staining of transfected myc-flag-tagged wild-type mouse CyAB in neural cells following transduction of CryAB R120G aggregate seeds. f Quantitative analysis of e . g Hypothetical model to explain the prion-like propagation of misfolded CryAB R120G protein from the heart to brain. Exosomes containing CryAB R120G protein can be transported from the heart to brain through blood circulation. It is also possible that CryAB R120G protein disrupts BBB and causes CryAB R120G crossing the BBB and entering the brain. In the brain, CryAB R120G protein propagates in a prion-like phenomenon, leading to disruption of BBB, increased neuroinflammation and abnormal brain function. Data are shown as mean ± SD; n = 6 for c and n = 3 for b, f . * p < 0.05, ** p < 0.01.

Article Snippet: Cells were transfected with myc-flag-CryAB plasmid (OriGene, #MR201515) encoding the myc-flag-tagged mouse CryAB protein.

Techniques: Incubation, Isolation, Cell Culture, Staining, Transfection, Transduction

Journal: Cell Stress & Chaperones

Article Title: Cell-free synthesis of functionally active HSPB5

doi: 10.1007/s12192-020-01073-5

Figure Lengend Snippet: Effect of magnesium ion concentration (a) and condensed S30 extract (b) on HSPB5 synthesis using the batch system. a HSPB5 was synthesized in a final volume of 50 μl using the batch system. Magnesium acetate (0.24 M) at different doses was added to each reaction mixture to achieve the indicated final concentration of Mg2+ (4.2 mM, 9.0 mM, 11.4 mM, or 13.8 mM). The Mg2+ concentration (4.2 mM) is carried over from 15 μl of the S30 extract. Aliquots from each reaction mixture were subjected to SDS-PAGE and Western blot analysis, as described in the “Materials and Methods.” For Western blot analysis, the X-ray film was exposed for 1 h for detection of signals. Right panel is the respective quantification graph of HSPB5’s intensity from the adjacent X-ray film. HSPB5 synthesis was dependent on the Mg2+ concentration. b Level of HSPB5 production compared between the S30 extract and condensed S30 extract. The Mg2+ concentration was adjusted as in Fig. 1a. The dose of S30 extract and condensed S30 extract was 15 μl. Upper blot: The X-ray film was exposed for 1 min rather than 1 h for detection of signals to avoid unmeasurable signal intensity derived from the condensed S30 extract due to overexposure. Lower blot: The X-ray film was exposed for longer time to detect signals from the S30 extract. Results of semi-quantification of HSPB5 synthesis by image analysis are illustrated to the right. Protein synthesis with condensed S30 extract resulted in higher HSPB5 production, as compared with the S30 extract, and the Mg2+ concentration was a crucial factor in protein synthesis for both systems

Article Snippet: The membrane was blocked for 2 h in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.0] 150 mM NaCl) containing 3% skim milk, washed with TBS, and then incubated overnight at 4 °C with an anti-rabbit polyclonal antibody against HSPB5 at a dilution of 1:3000 (Enzo Life Sciences, Farmingdale, NY, USA) in blocking solution.

Techniques: Concentration Assay, Synthesized, SDS Page, Western Blot, Derivative Assay

Journal: Cell Stress & Chaperones

Article Title: Cell-free synthesis of functionally active HSPB5

doi: 10.1007/s12192-020-01073-5

Figure Lengend Snippet: Effect of condensed S30 extract protein concentration on HSPB5 synthesis using the batch system. a Different doses of condensed S30 extract were added to the reaction mixture, and then HSPB5 was analyzed as described in the “Materials and Methods.” As shown in the right panel of a, with the high dose (5–15 μl) of condensed S30 extract, no obvious changes in protein production were observed; therefore, lower doses of condensed S30 extract were used to evaluate the dose dependence (see the left panel of a). The Mg2+ concentration was adjusted to 9.0 mM, as shown in Fig. ​Fig.1b,1b, because the Mg2+ concentration carried over from the condensed S30 extract varied in each reaction mixture. b Semi-quantification of HSPB5 synthesis by image analysis. DNA (−): complete reaction components except for HSPB5 cDNA. HSPB5 protein production was dependent on the dose of condensed S30 extract

Article Snippet: The membrane was blocked for 2 h in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.0] 150 mM NaCl) containing 3% skim milk, washed with TBS, and then incubated overnight at 4 °C with an anti-rabbit polyclonal antibody against HSPB5 at a dilution of 1:3000 (Enzo Life Sciences, Farmingdale, NY, USA) in blocking solution.

Techniques: Protein Concentration, Concentration Assay

Journal: Cell Stress & Chaperones

Article Title: Cell-free synthesis of functionally active HSPB5

doi: 10.1007/s12192-020-01073-5

Figure Lengend Snippet: Effect of different plasmid vectors on the efficiency of HSPB5 synthesis in the batch (a) and dialysis (b, c) systems. a HSPB5 cDNA was inserted into the pET14b vector and pIVEX2.4bNde vector, and protein synthesis using the batch system was carried out with condensed S30 extract (15 μl). The Mg2+ concentration was adjusted by addition of 0.24 M magnesium acetate, as indicated in Fig. ​Fig.1a.1a. Arrows show HSPB5 protein at lower and higher molecular weight derived from pET14b-HSPB5 and pIVEX2.4bNde-HSPB5 plasmid vectors, respectively, resulting from the different amino acid sequences in each vector for the His-tag region upstream of the HSPB5 cDNA. Arrowhead indicates non-specific signals on the blot membrane. X-ray film was exposed for 1 min for detection of signals. Use of the pIVEX-2.4bNde-HSPB5 plasmid vector produced a much larger amount of HSPB5 compared with use of the pET14b-HSPB5 plasmid vector. b The pIVEX2.4bNde vector was used for protein synthesis with condensed S30 extract, and the amount of HSPB5 synthesized in the batch and dialysis systems was compared. The dialysis system provides markedly higher HSPB5 synthesis. c Schematic illustration of the batch and dialysis systems

Article Snippet: The membrane was blocked for 2 h in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.0] 150 mM NaCl) containing 3% skim milk, washed with TBS, and then incubated overnight at 4 °C with an anti-rabbit polyclonal antibody against HSPB5 at a dilution of 1:3000 (Enzo Life Sciences, Farmingdale, NY, USA) in blocking solution.

Techniques: Plasmid Preparation, Concentration Assay, Molecular Weight, Derivative Assay, Produced, Synthesized

Journal: Cell Stress & Chaperones

Article Title: Cell-free synthesis of functionally active HSPB5

doi: 10.1007/s12192-020-01073-5

Figure Lengend Snippet: Effects of T7 RNA polymerase, phosphocreatine, PEG, and amino acid content in the reaction mixture on HSPB5 protein synthesis. a HSPB5 was synthesized using the dialysis system, and, after binding to a Ni+-NTA agarose beads column, HSPB5 was eluted with buffers of different pH containing 8 M urea. Aliquots were subjected to SDS-PAGE and Western blotting. Left and right panels show SDS-PAGE and Western blotting results, respectively. Arrow shows HSPB5 on Western blot. Eluates recovered using pH 4.5 buffer were stored as the HSPB5 fraction and used to evaluate the effect of different factors. b and c HSPB5 protein was synthesized under the conditions of 2-fold (X2) or 3-fold (X3) concentrations of T7 RNA polymerase (T7), phosphocreatine (PC), PEG8000 (PEG), and/or amino acids (AA), and then the amount of HSPB5 synthesized was compared with that obtained using the original components (Ori) described in the “Materials and Methods.” Aliquots of each reaction mixture before isolation with beads (c) and of each sample (b) prepared under denaturing conditions (8 M urea, pH 4.5 buffer) as shown in Fig. 4a were subjected to SDS-PAGE and Western blotting. The right panels of b and c show semi-quantification of each blot signal intensity

Article Snippet: The membrane was blocked for 2 h in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.0] 150 mM NaCl) containing 3% skim milk, washed with TBS, and then incubated overnight at 4 °C with an anti-rabbit polyclonal antibody against HSPB5 at a dilution of 1:3000 (Enzo Life Sciences, Farmingdale, NY, USA) in blocking solution.

Techniques: Synthesized, Binding Assay, SDS Page, Western Blot, Isolation

Journal: Cell Stress & Chaperones

Article Title: Cell-free synthesis of functionally active HSPB5

doi: 10.1007/s12192-020-01073-5

Figure Lengend Snippet: Purification (a), SDS-PAGE/Western blotting (b), and MS (c) of HSPB5 synthesized using the dialysis system. a HSPB5 synthesized using the dialysis system was isolated under native conditions. Isolation buffers containing a serial concentration of imidazole were used, and buffer containing 200 mM imidazole was chosen to purify HSPB5 after extensive washing with buffer containing 50 mM imidazole. b and c Aliquots of purified HSPB5 were subjected to SDS-PAGE and Western blotting (b) and MS (c)

Article Snippet: The membrane was blocked for 2 h in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.0] 150 mM NaCl) containing 3% skim milk, washed with TBS, and then incubated overnight at 4 °C with an anti-rabbit polyclonal antibody against HSPB5 at a dilution of 1:3000 (Enzo Life Sciences, Farmingdale, NY, USA) in blocking solution.

Techniques: Purification, SDS Page, Western Blot, Synthesized, Isolation, Concentration Assay

Journal: Cell Stress & Chaperones

Article Title: Cell-free synthesis of functionally active HSPB5

doi: 10.1007/s12192-020-01073-5

Figure Lengend Snippet: Inhibition of heat-induced aggregation of ADH and CS by HSPB5 synthesized using the batch and dialysis systems. Light scattering resulting from heat-induced aggregation of ADH and CS was assessed by measuring the increase in optical density at 360 nm, as described in the “Materials and Methods.” a HSPB5 prepared by the dialysis system exhibited dose-dependent inhibition of the increase in optical density associated with heat-induced aggregation of ADH. b HSPB5-D and HSPB5-B prepared by the dialysis and batch systems, respectively, showed similar inhibitory activity against heat–induced aggregation of ADH (left panel) and CS (right panel). In this study, heat-induced aggregation of ADH and CS was monitored at 39 °C and incubation time for ADH and CS were 30 and 45 min, respectively

Article Snippet: The membrane was blocked for 2 h in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.0] 150 mM NaCl) containing 3% skim milk, washed with TBS, and then incubated overnight at 4 °C with an anti-rabbit polyclonal antibody against HSPB5 at a dilution of 1:3000 (Enzo Life Sciences, Farmingdale, NY, USA) in blocking solution.

Techniques: Inhibition, Synthesized, Activity Assay, Incubation