ttbk2 sca11 polyclonal antibody  (Bioss)

Journal: The Journal of Cell Biology

Article Title: SARS-CoV-2 ORF10 impairs cilia by enhancing CUL2 ZYG11B activity

doi: 10.1083/jcb.202108015

Figure Lengend Snippet: Overlap of different expression proteins (DEPs) in ORF10 expressed and SARS-CoV-2 infected cells. (A) Principal component analysis (PCA) of proteome of control and ORF10 expression group. (B) Overlap between the DEPs in ORF10 expressed cells and previously identified SARS-CoV-2 infected proteome . (C) Number of proteins in both DEPs of ORF10 expressed cells and SARS-CoV-2 infected proteome. (D) A heatmap of downregulated proteins in ORF10 expressed cells and previously reported SARS-CoV-2 infected cells . (E) Overlap between the DEPs in ORF10 expressed cells and a published cilia-related proteome ( ; ; ). (F) The network of protein–protein interactions between ORF10-regulated cilium proteins according to the String database . (G) Overlap between the identified protein in this study and study. (H) Overlap between the DEPs in this study and study. (I) Immunoblotting analysis of the expression of pcDNA-ORF10-HA and pHAGE-ORF10-HA plasmids in transfected HEK293T cells within 24 h. (J) The protein level of TTBK2, BBS4, SEPTIN2, and IFT46 was dramatically decreased along with the gradient increased ORF10 expression. Immunoblotting analysis of TTBK2, BBS4, SEPTIN2, IFT46, and ORF10 levels in ORF10-expressing and empty vector (NC) control HEK293T cells. GAPDH served as a loading control. (K) Odyssey-based quantification of protein levels in J ( n = 3 independent experiments). Data are presented as the mean ± SEM. Statistical analysis was performed with two-tailed unpaired Student’s t test. (L) Cells transfected with pcDNA-ORF10-HA display reduced level of multiple cilium-related proteins. Immunoblotting analysis of TTBK2, BBS4, SEPTIN2, IFT46, and ORF10 levels in ORF10-expressing and empty vector (NC) control HEK293T cells. GAPDH served as a loading control. (M) Odyssey-based quantification of protein levels in L ( n = 3 independent experiments). Data are presented as the mean ± SEM. * indicates P < 0.05, ** indicates P < 0.01. Statistical analysis was performed with two-tailed unpaired Student’s t test. (N) Stability analysis of the SNX11 degron-fused EGFP with exogenous expression of ORF10 and ZYG11B proteins. overexpression of ZYG11B substantially reduced the EGFP/DsRed ratio by accelerated degradation of the SNX11 degron-fused EGFP protein. Source data are available for this figure: .

Article Snippet: Rabbit antibodies to TTBK2 (1:1,000, bs-11771R) and rabbit antibodies to Cytokeratin5 (1:200, bs-1208) were purchased from Bioss.

Techniques: Expressing, Infection, Western Blot, Transfection, Plasmid Preparation, Two Tailed Test, Over Expression

Journal: The Journal of Cell Biology

Article Title: SARS-CoV-2 ORF10 impairs cilia by enhancing CUL2 ZYG11B activity

doi: 10.1083/jcb.202108015

Figure Lengend Snippet: ORF10 downregulates cilium-related proteins. (A) Volcano plots of differentially expressed proteins (DEPs) were detected in a quantitative proteomic analysis of ORF10-expressing and empty vector control HEK293T cells. The red spots and blue spots indicate significantly upregulated and downregulated proteins, respectively. (B) Functional classification analysis of DEPs according to enrichment analysis of the biological process (BP) and cellular component (CC) terms and KEGG pathways. (C) The network of DEPs and enriched cilium-related gene ontology terms. (D) Cells expressing ORF10 display reduced accumulation of multiple cilium-related proteins. Immunoblotting analysis of TALPID3, TTBK2, BBS4, SEPTIN2, IFT46, and ORF10 levels in ORF10-expressing and empty vector (NC) control HEK293T cells. GAPDH served as a loading control. (E) Odyssey-based quantification of protein levels in D ( n = 3 independent experiments). Data are presented as the mean ± SEM. ** indicated P < 0.01, *** indicated P < 0.001. Statistical analysis was performed with two-tailed unpaired Student’s t test. Source data are available for this figure: .

Article Snippet: Rabbit antibodies to TTBK2 (1:1,000, bs-11771R) and rabbit antibodies to Cytokeratin5 (1:200, bs-1208) were purchased from Bioss.

Techniques: Expressing, Plasmid Preparation, Functional Assay, Western Blot, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: SARS-CoV-2 ORF10 impairs cilia by enhancing CUL2 ZYG11B activity

doi: 10.1083/jcb.202108015

Figure Lengend Snippet: ORF10 promotes IFT46 degradation by stimulating CUL2 ZYG11B activity. (A) ORF10 reduces TALPID3, TTBK2, BBS4, SEPTIN2, and IFT46 levels in a ubiquitin–proteasome pathway-dependent manner. Immunoblotting analysis of TALPID3, TTBK2, BBS4, SEPTIN2, IFT46, and ORF10 levels in ORF10-expressing and empty vector control HEK293T cells that were treated with or without the proteasome inhibitor MG-132 or the autophagy-lysosome degradation inhibitor 3-MA. GAPDH served as a loading control. (B) Odyssey-based quantification of protein levels in A ( n = 3 independent experiments). Data are presented as the mean ± SEM. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. Statistical analysis was performed with two-tailed unpaired Student’s t test. (C) ORF10 promotes the degradation of IFT46. A cycloheximide chase (CHX) assay of IFT46 was performed in ORF10-expressing and empty vector control HEK293T cells. Samples were taken at 0, 1, 2, and 4 h after the addition of CHX. The IFT46 and ORF10 protein levels were detected by immunoblotting using rabbit IFT46 antibody and rabbit HA antibody. GAPDH served as a loading control. (D) Quantification of the IFT46 levels in C. The protein level of IFT46 was quantified using Odyssey software. ( n = 3 independent experiments). Data are presented as the mean ± SEM. * indicates P < 0.05. Statistical analysis was performed with two-tailed unpaired Student’s t test. (E) ZYG11B physically interacts with IFT46. MYC-IFT46 and ZYG11B-FLAG (or empty vector) were co-transfected into HEK293T cells. 24 h after transfection, cells were collected for immunoprecipitation (IP) with an anti-MYC antibody, followed by analysis using anti-FLAG and anti-MYC antibodies. (F) CUL2 ZYG11B promotes the ubiquitination of IFT46. MYC-IFT46, ZYG11B-FLAG, and HA-Ub were transfected into HEK293T cells. 24 h after transfection, cells were collected for immunoprecipitation (IP) with an anti-MYC antibody, followed by analysis using anti-HA and anti-MYC antibodies. (G) The IFT46 level is dramatically decreased in ZYG11B-overexpressing HEK293T cells. Immunoblotting analysis against IFT46, FLAG, and HA for control, ORF10-expressing, and ZYG11B-overexpressing HEK293T cells. GAPDH served as a loading control. (H) Overexpression of ZYG11B promotes IFT46 degradation. A cycloheximide chase (CHX) assay of IFT46 was performed ZYG11B-overexpressing HEK293T cells. Samples were taken at 0, 1, 2, and 4 h after the addition of CHX. The IFT46 and ZYG11B protein levels were assessed with immunoblotting. GAPDH served as a loading control. (I) Quantification of the relative IFT46 levels in H. The protein level of IFT46 were quantified using Odyssey software. ( n = 3 independent experiments). Data are presented as the mean ± SEM. * indicates P < 0.05. Statistical analysis was performed with two-tailed unpaired Student’s t test. (J) ORF10 promotes the ubiquitination of IFT46. MYC-IFT46 and FLAG-Ub were transfected into HEK293T cells. 24 h after transfection, cells were collected and added with ORF10 or SARS-ORF10 for immunoprecipitation (IP) with an anti-MYC antibody, followed by analysis using anti-FLAG and anti-MYC antibodies. (K) IFT46 overexpression partially rescues the cilia biogenesis defects of ORF10-expressing NIH3T3 cells. Immunofluorescence analysis for Ac-Tubulin (green) and HA (red) in ORF10-expressing, ORF10/IFT46 co-transfected, and empty-vector cells. Nuclei were stained with DAPI (blue). (L) Distribution of ciliated cells among the ORF10-expressing, ORF10/IFT46 co-transfected, and empty-vector cells (K; n = 5 independent experiments). Data are presented as the mean ± SEM. ** indicates P < 0.01. Statistical analysis was performed with two-tailed unpaired Student’s t test. Source data are available for this figure: .

Article Snippet: Rabbit antibodies to TTBK2 (1:1,000, bs-11771R) and rabbit antibodies to Cytokeratin5 (1:200, bs-1208) were purchased from Bioss.

Techniques: Activity Assay, Western Blot, Expressing, Plasmid Preparation, Two Tailed Test, Software, Transfection, Immunoprecipitation, Over Expression, Immunofluorescence, Staining