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. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: J Immunol. 2017 Oct 20;199(11):3892–3899. doi: 10.4049/jimmunol.1700697

Figure 2. The long HLA-A 3’UTR results in less luciferase activity and protein expression as compared to the short form.

Figure 2

(A) Design of the luciferase reporter constructs used in this study is shown. The active PAS are indicated by black triangles, whereas disrupted PAS are indicated by open grey triangles. (B) The HLA-A 3’UTR luciferase constructs were transfected into Jurkat cell lines. Total RNA was extracted from the transfected cells after 18 hours. Specific primers were used to measure the abundance of Renilla and Firefly luciferase transcripts by qPCR. The normalized mRNA abundance is presented as a relative ratio Renilla/Firefly mRNA. The data represent triplicates in each experimental group. (C) Renilla and firefly luciferase activity was estimated by dual luciferase assays and presented as the normalized ratio of Renilla vs Firefly luciferase activity. The data represent six replicates in each group. The mean ±SE are depicted as horizontal and vertical bars for each group, respectively, and one of three comparable experiments that were performed is shown. Non-parametric Wilcoxon-Mann-Whitney tests were used for statistical comparisons and two tailed p values are indicated. (D) Design of the HLA-AORF-Flag constructs used in this study is shown. The active PAS are indicated by black triangles, whereas disrupted PAS are indicated by open grey triangles. (E) Jurkat cells were transfected with HLA-AORF-Flag plasmids. Total mRNA was extracted from the transfected cells, and cell lysates were extracted from the transfected cells after 24 hours. Specific primers were used to measure the abundance of HLA-A and Firefly luciferase transcripts by qPCR. The normalized mRNA abundance is presented as a relative ratio of HLA-A/Firefly mRNA. The data represent triplicates in each experimental group. (F) Total cell lysates of Jurkat cells transfected with HLA-AORF-Flag plasmids were used to carry out Western blot analysis with anti-Flag and anti-firefly luciferase antibodies. One of two independent experiments is shown.