primary antibody against cacybp sip  (Cell Signaling Technology Inc)

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Influence of CacyBP/SIP on α-synuclein aggregation. ( A, left panel ) Representative curves showing ThT fluorescence of 30 µM α-synuclein alone (black) or in the presence of: 30 µM inactive CacyBP/SIP (grey), 0.5 µM CacyBP/SIP (light green), 30 µM CacyBP/SIP (green) or 0.5 µM HSP90 (blue). ( A, right panel ) Statistical analysis of the results for samples taken on day 4 of incubation ( n = 3). ( B, left panel ) Dot-blots showing α-synuclein immunostaining in the soluble fraction (supernatant) in samples taken on day 4 of α-synuclein aggregation, alone or in the presence of 30 µM CacyBP/SIP and ( B, right panel ) densitometric analysis of the results ( n = 3). Data, calculated as means ± SEM, are presented as percentage values. * p ≤ 0.05, *** p ≤ 0.001.

Article Snippet: After overnight incubation with gentle agitation at 4 °C, wells were washed as above and primary antibody against CacyBP/SIP (Cell Signaling Technology, Danvers, MA, USA), diluted 1:4000 in PBS-T, was added.

Techniques: Fluorescence, Incubation, Immunostaining

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Effect of different concentrations of CacyBP/SIP on α-synuclein aggregation. ( A, left panel ) Representative curves showing ThT fluorescence of 30 µM α-synuclein alone (black) or in the presence of: 30 µM inactive CacyBP/SIP (grey) or 30 µM (dark green), 5 µM (middle green) or 1 µM CacyBP/SIP (light green). ( A, right panel ) Statistical analysis of the results for samples taken on day 4 of incubation ( n = 3). ( B, left panel ) Dot-blots showing α-synuclein immunostaining in the soluble fraction (supernatant) of samples taken on day 4 of incubation of α-synuclein alone or in the presence of different concentrations of CacyBP/SIP and ( B, right panel ) densitometric analysis of the results ( n = 3). Data, calculated as means ± SEM, are presented as percentage values. * p ≤ 0.05, *** p ≤ 0.001.

Article Snippet: After overnight incubation with gentle agitation at 4 °C, wells were washed as above and primary antibody against CacyBP/SIP (Cell Signaling Technology, Danvers, MA, USA), diluted 1:4000 in PBS-T, was added.

Techniques: Fluorescence, Incubation, Immunostaining

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Effect of CacyBP/SIP on later stages of α-synuclein aggregation. ( Left panel ) Representative curves showing ThT fluorescence of 30 µM α-synuclein alone (black) or in the presence of 30 µM: inactive CacyBP/SIP (grey) or CacyBP/SIP (green) added on day 4. ( Right panel ) Statistical analysis of the results of samples taken on day 6 ( n = 3). Data, calculated as means ± SEM, are presented as percentage values.

Article Snippet: After overnight incubation with gentle agitation at 4 °C, wells were washed as above and primary antibody against CacyBP/SIP (Cell Signaling Technology, Danvers, MA, USA), diluted 1:4000 in PBS-T, was added.

Techniques: Fluorescence

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Influence of CacyBP/SIP on α-synuclein aggregation assessed by transmission electron microscopy (TEM). Representative micrographs of α-synuclein obtained before initiation of aggregation ( A ), on day 4 of incubation ( B ), on day 4 of incubation in the presence of: 30 µM inactive CacyBP/SIP ( C ), 15 µM CacyBP/SIP ( D ) or 30 µM CacyBP/SIP ( E ). Scale bar—200 nm (A–D) and 100 nm ( E ).

Article Snippet: After overnight incubation with gentle agitation at 4 °C, wells were washed as above and primary antibody against CacyBP/SIP (Cell Signaling Technology, Danvers, MA, USA), diluted 1:4000 in PBS-T, was added.

Techniques: Transmission Assay, Electron Microscopy, Incubation

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Influence of CacyBP/SIP domains on α-synuclein aggregation. ( A, left panel ) Representative curves showing ThT fluorescence of 30 µM α-synuclein alone (black) or in the presence of 30 µM: inactive CacyBP/SIP (grey), CacyBP/SIP (green), N domain (orange), CS domain (yellow) or SGS domain (brown). ( A, right panel ) Statistical analysis of the results ( n = 3). ( A’, left panel ) Dot-blots showing α-synuclein immunostaining in the soluble fraction and ( A’, right panel ) statistical analysis of the results ( n = 3). (B, left panel) Representative curves showing ThT fluorescence of 30 µM α-synuclein alone (black) or in the presence of 30 µM: inactive CacyBP/SIP (grey), CacyBP/SIP (green) or NCS fragment of CacyBP/SIP (violet) ( B, right panel ) Statistical analysis of the results ( n = 3). ( B’, left panel ) Dot-blots showing α-synuclein immunostaining in the soluble fraction and ( B’, right panel ) statistical analysis of the results ( n = 3). In all cases, samples were taken on day 4 of incubation and data, calculated as means ± SEM, are presented as percentage values. ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: After overnight incubation with gentle agitation at 4 °C, wells were washed as above and primary antibody against CacyBP/SIP (Cell Signaling Technology, Danvers, MA, USA), diluted 1:4000 in PBS-T, was added.

Techniques: Fluorescence, Immunostaining, Incubation

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Interaction between α-synuclein and CacyBP/SIP studied by ELISA ( A ) and chemical crosslinking ( B ). ( A ) Upper panel shows absorbance measured at different molar ratio of CacyBP/SIP (concentration 0, 3.89, 7.78, 19.45, 31.12, and 58.35 µM) to α-synuclein (3.89 µM) while the lower one shows quantitative analysis of the results obtained from 3 independent experiments. Data are presented as means ± standard errors (SEM); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( B ) 15% polyacrylamide gel stained with Coomassie brilliant blue R250. α-synuclein (30 μM) was mixed with CacyBP/SIP (30 μM) Lane 1 and 4, α-synuclein alone; Lanes 2 and 5, mixture of α-synuclein and CacyBP/SIP; Lanes 3 and 6, CacyBP/SIP alone. Proteins were incubated with (lanes 1–3) and without crosslinking agent (lanes 4–6) and then 15 µl of reaction mixture was applied on the gel. “*” indicates the α-synuclein-CacyBP/SIP crosslinking product. A representative experiment, out of 3 performed, is shown.

Article Snippet: After overnight incubation with gentle agitation at 4 °C, wells were washed as above and primary antibody against CacyBP/SIP (Cell Signaling Technology, Danvers, MA, USA), diluted 1:4000 in PBS-T, was added.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Staining, Incubation

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Presence of complexes formed between α-synuclein and CacyBP/SIP or HSP90 in HEK293 cells visualized by PLA (representative images). Complexes of examined proteins are shown as red dots; cell nuclei, stained with DAPI, are in blue. HSP90 was used as a positive control. Scale bar–10 μm.

Article Snippet: After overnight incubation with gentle agitation at 4 °C, wells were washed as above and primary antibody against CacyBP/SIP (Cell Signaling Technology, Danvers, MA, USA), diluted 1:4000 in PBS-T, was added.

Techniques: Staining, Positive Control

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Influence of CacyBP/SIP on the number of α-synuclein aggregates in HEK293 cells. ( A ) A scheme showing preparation of α-synuclein seeds and their delivery to HEK293 cells. ( B, upper part ) Representative immunofluorescence staining performed with the use of primary conformation-specific anti-α-synuclein antibody. Aggregates/inclusions of α–synuclein are visible in green. Insert shows enlargement of inclusion. ( B, lower part ) Statistical analysis of the results from 3 independent experiments (30 inclusion-containing cells were analyzed) are presented as means ± standard errors (SEM); * p ≤ 0.05. Scale bar—5 μm.

Article Snippet: After overnight incubation with gentle agitation at 4 °C, wells were washed as above and primary antibody against CacyBP/SIP (Cell Signaling Technology, Danvers, MA, USA), diluted 1:4000 in PBS-T, was added.

Techniques: Immunofluorescence, Staining

Journal: Cells

Article Title: HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation

doi: 10.3390/cells9102254

Figure Lengend Snippet: Viability of HEK293 cells overexpressing CacyBP/SIP after rotenone treatment. Cells treated with solvent (black bar), cells transfected with 3xFLAG (white bar) or with 3xFLAG-CacyBP/SIP (grey bar) treated with rotenone. Cell viability was quantified using an MTS assay. Data obtained from 3 independent experiments, calculated as means ± SEM, are presented as percentage values. * p ≤ 0.05.

Article Snippet: After overnight incubation with gentle agitation at 4 °C, wells were washed as above and primary antibody against CacyBP/SIP (Cell Signaling Technology, Danvers, MA, USA), diluted 1:4000 in PBS-T, was added.

Techniques: Transfection, MTS Assay