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. 2017 Nov 21;8:1640. doi: 10.1038/s41467-017-01601-5

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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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Fig. 6 — Impaired antigen degradation and cross-presentation in cells with STIM1 ablation. a Quantitative PCR analysis of Stim1 and Stim2 gene expression in DCs nucleofected with Stim1-specific siRNA (mean ± S.E.M.; n = 3 experiments). b Immunoblot of STIM1 and actin expression in WT (control siRNA) and Stim1-silenced (siRNA #5) DCs and quantification of actin/STIM1 ratio of three independent experiments (**P < 0.01 via unpaired t-test). c Averaged Ca²⁺ signals in control and STIM1-silenced DCs loaded with fluo-4AM and stimulated with TG in Ca²⁺-free solution before adding 2 mM Ca²⁺. One representative experiment is shown (left panel) (n = 3 × 10⁵ cells) and graph shows Ca²⁺ influx (right) from three independent experiments normalized to siRNA control cells (****P < 0.0001 via paired t-test). d Proteolysis was measured by quantifying the degradation of OVA in control and STIM1-silenced phagosomes for the indicated time points (n = 3 experiments; mean ± S.E.M.; *P < 0.05, ***P < 0.001 via paired t-test). e Endosomal pH in control and STIM1-silenced DCs pulsed for 10 min with FITC-coupled and Alexa-647-coupled dextrans and chased for 50 min (n = 3; mean ± S.E.M.). f DCs knocked down for STIM1 or not from WT and 3d/3d mice were challenged with BSAb, OVAb, or OVA-transfected splenocytes as sources of exogenous antigen before co-culture with CFSE-labeled OT-I T cells. T cell proliferation was monitored by flow cytometry 3 days later. Representative histogram plots are shown (left). Quantification (right) of OT-I cell division is shown as mean percentage of proliferating cells (n = 3; mean ± S.E.M.; *P < 0.05; **P < 0.01; ****P < 0.0001 using unpaired t-test). g Immunoblot analysis of STIM1 and actin protein expression in control human fibroblasts and fibroblasts from a patient with STIM1 deficiency and quantification of actin/STIM1 ratio. ****P < 0.01; n = 3 (unpaired t-test). h Control and STIM1-deficient FcγRIIA-EGFP-K^b-transduced human fibroblasts were stimulated with precipitated OVA immunocomplexes (OVA pICs) or BSA pICs (none) as control (upper panel) or SIINFEKL peptide (lower panel) before co-culture with B3Z hybridoma. IL-2 secretion was measured by ELISA (n = 3; mean ± S.E.M.; ***P < 0.001 via unpaired t-test)