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. 2017 Jul 19;7:5941. doi: 10.1038/s41598-017-05734-x

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© The Author(s) 2017

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/.

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SREBP expression and processing in the absence of calreticulin. (A) Q-PCR quantitative analysis of total SREBP-1 and SREBP-2 mRNA abundance in wild-type and Calr ^−/− cells. Results were normalized to 18S rRNA (internal control). NS, not significant (Student’s t-test). Representative of 5 biological replicates. (B) Immunoblot analysis of SREBP-1, nSREBP-1, SREBP-2 and nSREBP-2 protein in wild-type and Calr ^−/− cells. Anti-γ-tubulin antibodies were used as a loading control. Representative of 3 biological replicates. (C,D) Quantitative analysis of immunoblots showing the ratio of nuclear to total SREBP-1 (C) and total SREBP-2 (D) in wild-type and Calr ^−/− cells. The value for the total is the sum of precursor and nuclear forms of SREBP. *Indicates statistically significant differences: SREBP-1, p-value = 0.0017 (Student’s t-test); SREBP-2, p-value = 0.0218 (Student’s t-test). Representative of 3 biological replicates. (E) GFP:SBP-1 accumulation in the intestinal nucleus (arrows) in wild-type N2 and calreticulin deficient crt-1 C. elegans. Worms expressing GFP:SBP-1 driven by sbp-1 promoter are shown. The average ratio of fluorescence in the nucleus and cytoplasm was calculated in each worm, and scatter-plotted (right panel). **Indicates statistically significant differences: p-value < 0.01 (Student’s t-test). Representative of 3 biological replicates. (F) Wild-type, Calr ^−/− cells and Calr ^−/− cells transfected with a calreticulin expression vector (Calr ^−/− +rCalr) were co-transfected with the SRE luciferase reporter plasmid followed by luciferase assay. *Indicates statistically significant difference: p-value = 0.0006 (Student’s t-test). Representative of 3 biological replicates. rCalr, recombinant calreticulin. Inset: immunoblot analysis with anti-Calr antibodies. (G) HEK293T cells were transfected with calreticulin specific siRNA or scrambled siRNA and with the SRE luciferase reporter plasmid. Representative of 3 biological replicates. Inset: immunoblot analysis with anti-Calr antibodies. Calr, calreticulin. Ctrl, control. The images of (B) shown are cropped. The full-length gels/blots are shown in Fig. S8. See “Experimental Procedures” for additional details.