Fig 1: IL-6 secretion by RNA1.2 mutant-infected cells after NF-κB inhibition or TPRG1L knockdown. (A) HFFF2 cells were mock-infected or infected with WT, ΔRNA1.2 or ΔTATA at MOI = 5. At 72 h p.i., the cells were incubated with fresh medium lacking (NT) or containing IKKα-and-IKKβ inhibitor BMS-345541 (IKKi) for a further 6 h. The medium was harvested, and secreted IL-6 was analyzed by ELISA. (B) Immortalized HFT cells were transduced with lentiviruses encoding a non-targeting shRNA negative control (Neg) or a TPRG1L-targeting shRNA (KD). After the establishment of stably expressing cell lines, the cells were infected with WT, ΔRNA1.2 or ΔTATA at MOI = 5. The cells were incubated with fresh medium between 72 and 78 h p.i., which was then harvested and analyzed for secreted IL-6 by ELISA. RNA was also isolated at 78 h p.i. and analyzed by RT-qPCR in (C). (C) Expression of TPRG1L mRNA in knockdown samples calculated relative to that of Neg samples. Data from four independent experiments are shown in each of (A–C). The error bars denote standard error values, and significant differences are marked by asterisks (p < 0.003, paired t-test; p < 0.05, Wilcoxon matched-pairs signed rank test). (D) MCP-1 and CXCL1 transcript levels were monitored during infection of HFFF2 cells with WT, ΔRNA1.2 or ΔTATA at MOI = 5. At 72 h p.i., the cells were incubated with fresh medium lacking or containing TNF-α for an additional 6 h. RNA was then harvested and analyzed by RT-PCR. The data are representative of three experiments.
Fig 2: TPRG1L transcript and protein levels in RNA1.2 mutant-infected cells. HFFF2 cells were infected with WT, ΔRNA1.2, or ΔTATA at MOI = 5. At 72 h p.i., RNA and protein were harvested and analyzed. (A) RT-qPCR data from three independent experiments, each with duplicates. Standard errors are marked by error bars. Significant differences are marked by asterisks (p < 0.001, paired t-test; p < 0.05, Wilcoxon matched-pairs signed rank test). (B) Immunoblotting data from one of three independent experiments.
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