Journal: PLoS ONE

Article Title: A screening system for identifying interacting proteins using biomolecular fluorescence complementation and transposon gene trap

doi: 10.1371/journal.pone.0251240

Figure Lengend Snippet: Antibody list.

Article Snippet: Anti-CACYBP rabbit mAb (D43G11) , #8225S , Cell signaling.

Techniques:

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: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

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: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

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: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

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: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

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: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

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: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

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: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

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: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

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: The membrane was subsequently incubated in TBS-T containing 5% skim milk for 1 h. After that, the primary rabbit polyclonal anti-CacyBP/SIP antibody (Cell Signaling Technology), diluted 1:1000, or mouse anti-α-synuclein antibody (Abcam) diluted 1:1000 was applied and incubation was carried out overnight at 4 °C.

Techniques: Transfection, MTS Assay

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

Journal: Experimental and Therapeutic Medicine

Article Title: CacyBP/SIP promotes tumor progression by regulating apoptosis and arresting the cell cycle in osteosarcoma

doi: 10.3892/etm.2020.8843

Figure Lengend Snippet: Lentivirus-mediated shRNA effectively decreases CacyBP/SIP expression in Saos-2 cells. (A) The levels of CacyBP/SIP expression in a variety of osteosarcoma cell lines. After 3 days of CacyBP/SIP lentiviral transfection, the transfection efficiency was (B) observed with fluorescence and (C) determined with reverse transcription-quantitative PCR. Magnification, x100. (D) Decreased CacyBP/SIP protein level was detected in CacyBP/SIP-shRNA lentivirus-transfected Saos-2 cells. ** P<0.01. sh, short hairpin RNA; CacyBP/SIP, calcyclin-binding protein/Siah-1-interacting protein; Ctrl, control.

Article Snippet: Protein samples (20 µg) were loaded and electrophoresed in an SDS-PAGE (10% gel) at 120 mA for 1 h and subsequently transferred to PVDF membranes (EMD Millipore) at 300 mA for 120 min. After being blocked with TBS with Tween-20 (TBST) containing 5% (w/v) non-fat dried skim milk powder for 24 h at 4˚C, membranes were incubated with a CacyBP/SIP antibody (cat. no. 3354; 1:1,000) or a GAPDH antibody (cat. no. 3683; 1:1,000; both from Cell Signaling Technology, Inc.) overnight at 4˚C. p21 antibody (ab188224; 1:1,000), cyclin-dependent kinase (CDK) 2 antibody (ab32147; 1:1,000), CDK4 antibody (ab199728; 1:2,000), Cyclin D1 antibody (ab226977; 1:2,000), Cyclin E1 antibody (ab33911; 1:2,000), Bax antibody (ab32503; 1:2,000) and Bcl-2 antibody (ab692; 1:500) were purchased from Abcam.

Techniques: shRNA, Expressing, Transfection, Fluorescence, Real-time Polymerase Chain Reaction, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: CacyBP/SIP promotes tumor progression by regulating apoptosis and arresting the cell cycle in osteosarcoma

doi: 10.3892/etm.2020.8843

Figure Lengend Snippet: Knockdown of CacyBP/SIP inhibits Saos-2 cell proliferation and colony formation in vitro . Saos-2 cells expressing either CacyBP/SIP-shRNA lentivirus or Ctrl lentivirus were seeded in 96-well and 6-well plates and cell growth was monitored by a Celigo Imaging Cytometer daily for 5 days. The (A) total number of cells and (B) cell growth rate were determined. Magnification, x40. (C) MTT assay and (D) colony formation assay was performed to evaluate cell proliferation. Magnification, x100. (E) Statistical analysis of colony numbers between two groups. ** P<0.01. CacyBP/SIP, calcyclin-binding protein/Siah-1-interacting protein; sh, short hairpin RNA; Ctrl, control.

Article Snippet: Protein samples (20 µg) were loaded and electrophoresed in an SDS-PAGE (10% gel) at 120 mA for 1 h and subsequently transferred to PVDF membranes (EMD Millipore) at 300 mA for 120 min. After being blocked with TBS with Tween-20 (TBST) containing 5% (w/v) non-fat dried skim milk powder for 24 h at 4˚C, membranes were incubated with a CacyBP/SIP antibody (cat. no. 3354; 1:1,000) or a GAPDH antibody (cat. no. 3683; 1:1,000; both from Cell Signaling Technology, Inc.) overnight at 4˚C. p21 antibody (ab188224; 1:1,000), cyclin-dependent kinase (CDK) 2 antibody (ab32147; 1:1,000), CDK4 antibody (ab199728; 1:2,000), Cyclin D1 antibody (ab226977; 1:2,000), Cyclin E1 antibody (ab33911; 1:2,000), Bax antibody (ab32503; 1:2,000) and Bcl-2 antibody (ab692; 1:500) were purchased from Abcam.

Techniques: In Vitro, Expressing, shRNA, Imaging, Cytometry, MTT Assay, Colony Assay, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: CacyBP/SIP promotes tumor progression by regulating apoptosis and arresting the cell cycle in osteosarcoma

doi: 10.3892/etm.2020.8843

Figure Lengend Snippet: Knockdown of CacyBP/SIP induces Saos-2 cell apoptosis and cell cycle arrest. (A and B) The percentage of apoptotic cells was determined with Annexin V-APC. (C and D) Flow cytometry was performed to evaluate G 1 /S arrest in Saos-2 cells following knockdown of CacyBP/SIP. ** P<0.01. CacyBP/SIP, calcyclin-binding protein/Siah-1-interacting protein; sh, short hairpin RNA; Ctrl, control; OD, optical density.

Article Snippet: Protein samples (20 µg) were loaded and electrophoresed in an SDS-PAGE (10% gel) at 120 mA for 1 h and subsequently transferred to PVDF membranes (EMD Millipore) at 300 mA for 120 min. After being blocked with TBS with Tween-20 (TBST) containing 5% (w/v) non-fat dried skim milk powder for 24 h at 4˚C, membranes were incubated with a CacyBP/SIP antibody (cat. no. 3354; 1:1,000) or a GAPDH antibody (cat. no. 3683; 1:1,000; both from Cell Signaling Technology, Inc.) overnight at 4˚C. p21 antibody (ab188224; 1:1,000), cyclin-dependent kinase (CDK) 2 antibody (ab32147; 1:1,000), CDK4 antibody (ab199728; 1:2,000), Cyclin D1 antibody (ab226977; 1:2,000), Cyclin E1 antibody (ab33911; 1:2,000), Bax antibody (ab32503; 1:2,000) and Bcl-2 antibody (ab692; 1:500) were purchased from Abcam.

Techniques: Flow Cytometry, Binding Assay, shRNA

Journal: Experimental and Therapeutic Medicine

Article Title: CacyBP/SIP promotes tumor progression by regulating apoptosis and arresting the cell cycle in osteosarcoma

doi: 10.3892/etm.2020.8843

Figure Lengend Snippet: Prognosis analysis based on TCGA data. TCGA database analysis demonstrated that patients with high CacyBP/SIP mRNA levels demonstrated worse prognosis compared with patients with low CacyBP/SIP mRNA levels. (A) Disease free survival analysis. (B) Overall survival analysis. TCGA, The Cancer Genome Atlas; CacyBP/SIP, calcyclin-binding protein/Siah-1-interacting protein; sh, short hairpin RNA; Ctrl, control.

Article Snippet: Protein samples (20 µg) were loaded and electrophoresed in an SDS-PAGE (10% gel) at 120 mA for 1 h and subsequently transferred to PVDF membranes (EMD Millipore) at 300 mA for 120 min. After being blocked with TBS with Tween-20 (TBST) containing 5% (w/v) non-fat dried skim milk powder for 24 h at 4˚C, membranes were incubated with a CacyBP/SIP antibody (cat. no. 3354; 1:1,000) or a GAPDH antibody (cat. no. 3683; 1:1,000; both from Cell Signaling Technology, Inc.) overnight at 4˚C. p21 antibody (ab188224; 1:1,000), cyclin-dependent kinase (CDK) 2 antibody (ab32147; 1:1,000), CDK4 antibody (ab199728; 1:2,000), Cyclin D1 antibody (ab226977; 1:2,000), Cyclin E1 antibody (ab33911; 1:2,000), Bax antibody (ab32503; 1:2,000) and Bcl-2 antibody (ab692; 1:500) were purchased from Abcam.

Techniques: Binding Assay, shRNA