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. 2020 Apr 20;48(9):4725–4740. doi: 10.1093/nar/gkaa256

Figure 1.

Figure 1.

Experimental setup and cell fractionation for compartment-specific eRIC from steady state and stressed cells. (A) A schematic representation of the experimental strategy. HuH7 cells under steady state conditions or following treatment with arsenite were subjected to UV-crosslinking (CL) or not (noCL), and processed to generate clean cytoplasmic (C) and nuclear (N) fractions prior to compartment-specific eRIC. (B) HuH7 cells grown under steady state conditions or exposed to 100 μM Na-arsenite were fixed and immuno-stained against ELAVL1 (green). Cytoplasmic aggregation of ELAVL1 positively controls for stress induction. Nuclear counterstain is shown in blue. (C) Nuclear (blue) and ER (red) staining showing nuclear integrity and removal of ER by cell fractionation (left panel). For a negative control of ER removal, NP-40 in the lysis buffer was replaced with the less effective Tween-20 (right panel). (D) Analysis of cell fractionation. Equal aliquots of cell fractions were separated by SDS-PAGE and analyzed by immunoblotting against the indicated marker proteins.