Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: CIB1 and CHIP specifically interact. A SDS-PAGE gel of proteins bound to IgG (left lane) or CIB1 (right lane). Protein marker stands stand for (from top to bottom): 170, 130, 100, 70 (Red), 55, 40, 35, 25, 15/10 kDa. B Endogenous interaction between CIB1 and CHIP in PC-9 cells. C PC-9 cells were transiently transfected for 36 h with CHIP plasmid, and cell lysates were immunoblotted with indicated antibodies. D HEK293 cells were transiently transfected for 36 h with Flag-CIB1 and Myc-CHIP, and cell lysates were immunoblotted with indicated antibodies. E Representative immunohistochemically stained images of LAC tissues using the anti-CHIP and anti-CIB1 antibodies. Areas in the black squares are magnified in the right slide panels. F Pearson analysis correlation between CIB1 and CHIP expression in LAC tissues ( r = −0.3582, P = 0.0049) G HEK293 cells were transfected with Flag-CIB1 (1 μg) and various concentrations of Myc-CHIP plasmids (0, 0.2, 0.5, and 1 μg). And after 36 h, cell lysates were immunoblotted with anti-Flag, anti-Myc, and anti-Tublin antibodies. H Representative confocal images of immunostaining for CHIP (green) and CIB1 (red) in PC-9 and A549 cells. Scale bar, 50 μm. I Cells were treated with 40 μg/ml cycloheximide for the indicated times, and cell lysates were immunoblotted with indicated antibody. J HEK293 cells were transfected for 36 h with Flag-CIB1 alone or together with Myc-CHIP and treated with MG-132 (10 μM), or leupeptin (100 μM) for 4 h. Cell lysates were analyzed by western blotting with the indicated antibodies.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: SDS Page, Marker, Transfection, Plasmid Preparation, Staining, Expressing, Immunostaining, Western Blot

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A PC-9 were transfected with CHIP plasmid for 36 h. Cell extracts were immunoprecipitated with the anti-CIB1 antibody, followed by immunoblotting with the anti-Ubiquitin antibody. B HEK293 cells were transfected with HA-ubiquitin, Flag-CIB1 with or without Myc-CHIP. Cell lysates were immunoprecipitated with the anti-HA antibody, followed by immunoblotting with anti-flag antibodies. C PC-9 cells were transfected with CHIP or CHIP-ΔUbox plasmid. Cell extracts were immunoprecipitated with the anti-ubiquitin antibody, followed by immunoblotting with the anti-CIB1 antibody. D HEK293 cell were transfected with Flag-CIB1, HA-ubiquitin, with or without Myc-CHIP or Myc -CHIP-ΔUbox. Denatured cell lysates were immunoprecipitated with the anti-HA antibody, followed by immunoblotting with Flag antibodies. E Where specified, purified E1, E2, ubiquitin, CHIP and CIB1 proteins were incubated with in vitro ubiquitination buffers. Reaction samples were analyzed by SDS-polyacrylamide gel electrophoresis, followed by Sliver staining or immunoblotting with the indicated antibodies. F HEK293 cells were transfected for 36 h with Flag-CIB1, Myc-CHIP, HA-Ubiquitin or HA-K48R-ubiquitin or HA-K63R-ubiquitin. Cell extracts were immunoprecipitated with the anti-HA antibody followed by immunoblotting with the anti-Flag antibody.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Purification, Incubation, In Vitro, Polyacrylamide Gel Electrophoresis, Staining

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A HEK293 cells were co-transfected with HA-ubiquitin and Flag-CIB1 and its corresponding mutated plasmids for 36 h. Cell lysates were subjected to immunoprecipitation with HA antibody, followed by Flag immunoblotting. B HEK293 cells were co-transfected with HA-ubiquitin with HA-ubiquitin, and Flag-CIB1 or Flag-K10R-CIB1 or Flag-K65R-CIB1 or Flag-K10R+K65R-CIB1. Cell lysates were subjected to immunoprecipitation with HA antibody, followed by Flag immunoblotting. C HEK293 cells were co-transfected with Flag-CIB1, Flag-K10R-CIB1, Flag-K65R-CIB1, Flag-K10R+K65R-CIB1 and Myc-CHIP. Cell lysates were analyzed by immunoblotting with MYC, Flag and α-Tublin antibodies. D Computational modeling results show multiple binding sites for ubiquitin on CIB1. The red region is the binding site for CIB1.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A , B Representative images and quantification of the Transwell assays using transfected A549/H1299 cells. C , D Quantification of the wound healing assays using transfected A549/H1299 cells. E Western blot assay showing the phosphorylation of AKT and EMT enhanced by CIB1. Restoration of CHIP attenuated this elevation in LAC cells.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Transfection, Western Blot

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A Representative luciferase images and quantification of average luciferase intensity of lungs in the i.v. metastasis assay. B , C Representative photographs and quantification of metastatic tumor nodes in mouse lungs from the i.v. metastasis assay. D Representative immunohistochemically stained images of lung tissue using anti-CIB1, anti-E-cadherin, and N-cadherin antibody. E CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal and tumor metastasis in lung adenocarcinoma through AKT pathway. * P < 0.05 vs negative control (NC) group, ** P < 0.01 vs NC group.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Luciferase, Staining, Negative Control