Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2018 Aug 28;26(6):1062–1076. doi: 10.1038/s41418-018-0185-5

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 7 — The C-terminal cleavage fragment of CALCOCO2 retains the function of full-length CALCOCO2 in promoting CVB3 growth. a, b HeLa cells were transfected with 3×FLAG-tagged CALCOCO2^WT, N- or C-terminal cleavage fragments of CALCOCO2 (N-CALCOCO2 or C-CALCOCO2) in the presence of vehicle (DMSO), proteosomal inhibitor (MG132, 10 µM) (a), or lysosome inhibitor (bafilomycin, 200 nM) (b) for 6 h. Protein levels of full length and the respective cleavage fragments of CALCOCO2 were verified by western blotting with an anti-Flag antibody. Ubiquitin (a) and LC3 (b) were probed by western blotting to confirm the inhibition of proteasome and lysosome activities, respectively. Densitometry was conducted as in Fig. 1. c HeLa cells were transfected with 3×FLAG-tagged CALCOCO2^WT, C-CALCOCO2, or empty vector for 24 h, followed by CVB3 infection (MOI = 10) for 7 h. Virus titer (mean ± SD, n = 3) were measured by TCID₅₀ and western blotting was performed to confirm the expression of exogenous CALCOCO2 using an anti-Flag antibody. d Knockout (KO) efficiency of the CALCOCO2 KO HeLa cells was verified by western blotting with anti-CALCOCO2 antibody. e CALCOCO2 KO cells were transfected with empty vector, 3×FLAG-tagged CALCOCO2^WT or C-CALCOCO2 for 16 h. Western blot analysis was conducted to examine the protein expression of MAVS, Flag-CALCOCO2, and ACTB. Protein levels of MAVS were quantitated by densitometric analysis, normalized to ACTB, and presented as fold changes (mean ± SD, n = 3) compared with vector control. f, g HeLa cells were co-transfected with empty vector, 3×FLAG-tagged CALCOCO2^WT or C-CALCOCO2, together with either GFP-LC3 (f) or HA-Ubiquitin (g) for 16 h. Co-IP and western blotting were conducted as above. Blots for IgG heavy chain and ACTB were used as loading controls for IP and input, respectively. Results represent data from two independent experiments. h, i CALCOCO2 KO HeLa cells were transfected with empty vector, 3×FLAG-tagged CALCOCO2^WT or C-CALCOCO2 for 16 h, followed by addition of poly I:C (1 μg/ml) for another 12 h. Cells were collected for RNA extraction and qRT-PCR was conducted to determine the mRNA levels of IFN-β (H, mean ± SD, n = 3). Culture supernatants were harvested for the measurement of IFN-β secretion by ELISA (I, mean ± SD, n = 3)

Fig. 7 — The C-terminal cleavage fragment of CALCOCO2 retains the function of full-length CALCOCO2 in promoting CVB3 growth. a, b HeLa cells were transfected with 3×FLAG-tagged CALCOCO2^WT, N- or C-terminal cleavage fragments of CALCOCO2 (N-CALCOCO2 or C-CALCOCO2) in the presence of vehicle (DMSO), proteosomal inhibitor (MG132, 10 µM) (a), or lysosome inhibitor (bafilomycin, 200 nM) (b) for 6 h. Protein levels of full length and the respective cleavage fragments of CALCOCO2 were verified by western blotting with an anti-Flag antibody. Ubiquitin (a) and LC3 (b) were probed by western blotting to confirm the inhibition of proteasome and lysosome activities, respectively. Densitometry was conducted as in Fig. 1. c HeLa cells were transfected with 3×FLAG-tagged CALCOCO2^WT, C-CALCOCO2, or empty vector for 24 h, followed by CVB3 infection (MOI = 10) for 7 h. Virus titer (mean ± SD, n = 3) were measured by TCID₅₀ and western blotting was performed to confirm the expression of exogenous CALCOCO2 using an anti-Flag antibody. d Knockout (KO) efficiency of the CALCOCO2 KO HeLa cells was verified by western blotting with anti-CALCOCO2 antibody. e CALCOCO2 KO cells were transfected with empty vector, 3×FLAG-tagged CALCOCO2^WT or C-CALCOCO2 for 16 h. Western blot analysis was conducted to examine the protein expression of MAVS, Flag-CALCOCO2, and ACTB. Protein levels of MAVS were quantitated by densitometric analysis, normalized to ACTB, and presented as fold changes (mean ± SD, n = 3) compared with vector control. f, g HeLa cells were co-transfected with empty vector, 3×FLAG-tagged CALCOCO2^WT or C-CALCOCO2, together with either GFP-LC3 (f) or HA-Ubiquitin (g) for 16 h. Co-IP and western blotting were conducted as above. Blots for IgG heavy chain and ACTB were used as loading controls for IP and input, respectively. Results represent data from two independent experiments. h, i CALCOCO2 KO HeLa cells were transfected with empty vector, 3×FLAG-tagged CALCOCO2^WT or C-CALCOCO2 for 16 h, followed by addition of poly I:C (1 μg/ml) for another 12 h. Cells were collected for RNA extraction and qRT-PCR was conducted to determine the mRNA levels of IFN-β (H, mean ± SD, n = 3). Culture supernatants were harvested for the measurement of IFN-β secretion by ELISA (I, mean ± SD, n = 3)