Fig 1: Multiple K residues of CIB1 are polyubiquitinated by CHIP.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 a-Tublin antibodies. D Computational modeling results show multiple binding sites for ubiquitin on CIB1. The red region is the binding site for CIB1.
Fig 2: CHIP upregulation alleviates CIB1 induced LAC migratory capacity and metastatic ability in vivo.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.
Fig 3: CHIP upregulation alleviates CIB1 induced LAC migratory capacity and metastatic ability in vitro.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.
Fig 4: CIB1 is frequently upregulated in LAC tissue and cell lines and associated with poor clinicopathological features and prognosis.A Representative statistics analysis of CIB1 levels in two different data using the ONCOMINE database (www.oncomine.org). Data were filtered to display upregulation of CIB1 in LAC tissues compared to normal tissues (T: Tumor, N: normal tissues). a: Hou Lung Statistics; b: Landi Lung Statistics. B Representative immunohistochemically stained images of normal and LAC tissue using anti-CIB1 antibody: a, b: negative expression of CIB1 in normal lung tissue; c, d: negative expression of CIB1 in lung adenocarcinoma; e, f: weakly positive expression of CIB1 in lung adenocarcinoma; g, h: moderately positive expression of CIB1 in lung adenocarcinoma; i, j: Strong positive expression of CIB1 in lung adenocarcinoma;. C Relative mRNA expression level of CIB1 in LAC tumor tissue compare to normal tissues from 60 patients. D Quantitative RT-PCR results showed CIB1 expression levels in five LAC cell lines and normal bronchus cell lines. E Western blots results showed CIB1 expression levels in five LAC cell lines and normal bronchus cell lines. F, G Metastatic or advanced TNM stage LAC tumors had higher CIB1 expression levels compared with those of nonmetastatic or early TNM stage LAC tumors. H, I Kaplan–Meier analysis showed that the 5-year overall survival and disease-free survival for LAC patients with higher CIB1 expression levels were significantly shorter than those of patients with lower CIB1 expression level. *P < 0.05; **P < 0.01; ***P < 0.001; HR, hazard ratio.
Fig 5: CHIP facilitates K48-linked polyubiquitination of CIB1.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-?Ubox 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-?Ubox. 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.
Supplier Page from Abcam for Anti-CIB1/KIP antibody