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. 2020 Mar 16;9:e52241. doi: 10.7554/eLife.52241

Figure 2. NF-κB c-Rel is a novel factor in the adaptive response to hyperosmotic stress.

Figure 2.

(A) Control and shPACT MEFs were treated with 500 or 600 mOsm. Nuclear fractions were isolated, and protein levels were analyzed via western blot. Insert indicates efficiency of PACT depletion from cytoplasmic extracts. (B) Control and shc-Rel MEFs were treated with 500 mOsm sucrose. Lysates were assayed for caspase-3 enzymatic activity. (C) Control and shc-Rel MEFs were treated with 500 or 600 mOsm sucrose. Nuclear fractions were isolated and protein levels were analyzed via western blot. (D) Control and shc-Rel MEFs were treated with 500 or 600 mOsm sucrose. RNA was isolated, and mRNA transcripts were analyzed via RT-qPCR. (E) TonEBP WT and TonEBP KO MEFs were treated with 500 mOsm sucrose. RNA was isolated, and mRNA transcripts were analyzed via RT-qPCR. (F) Control and shc-Rel MEFs were treated with varying intensities of sucrose. RNA was isolated, and mRNA transcripts were analyzed via RT-qPCR. (G) After indicated treatments, cytoplasmic membrane fractions were isolated and analyzed via western blot. (H) After indicated treatments, total lysates were isolated and analyzed via western blot. (I) Control and shc-Rel MEFs were treated with 500 mOsm sucrose. Intracellular levels of the amino acid proline were analyzed via amino acid uptake assay. (J) Control and shc-Rel MEFs and TonEBP WT and KO MEFs were transfected with the Slc38a2-promoter luciferase reporter construct, then treated with 500 mOsm sucrose. Luciferase activity was normalized to co-transfected Renilla luciferase activity. (K) Control and shc-Rel MEFs and TonEBP WT and KO MEFs were transfected with the Ppp1r15a-promoter luciferase reporter construct, then treated with 500 mOsm sucrose. Luciferase activity was normalized to co-transfected Renilla luciferase activity.

Figure 2—source data 1. Graph values for caspase-3 activity assays and RT-qPCR experiments in Figure 2.