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. 2019 Feb 13;47(5):2487–2505. doi: 10.1093/nar/gkz086

Figure 5.

Figure 5.

WDR79 is not involved in CB localization of UG-rich motif-bearing C/D scaRNAs. (A) Isolation of WDR79−TDP-43 complex by two-step pull-down (PD) method. DAP-TDP-43 complexes were first pulled down (1st-PD) with anti-FLAG from DAP-TDP-43-expressing T-REx 293 cells (induced with doxycycline for 24 h) and then pulled down (2nd-PD) with anti-WDR79 (protocol is shown at left). Protein components were detected by western blot analysis (IB) with anti-WDR79 or streptavidin-HRP (biotin). RNAs were detected by northern blot analysis (NB) with DNA probes complementary to the RNAs indicated on the right. Input consisted of 10 μg of cell lysate or 4 μg of RNA extracted from cell lysates. (B) HEF-WDR79-expressing T-REx 293 cells were treated with ncRNA (nc) or TDP-43 siRNA (si) for 72 h and further treated with Dox for 24 h. RNAs and proteins associated with HEF-WDR79 were immunoprecipitated with anti-FLAG antibody (IP: FLAG). Protein components were detected by western blot analysis (IB), and RNAs were by northern blot analysis (NB) with DNA probes complementary to the RNAs indicated on the right. Input, 10 μg of cell lysate or 4 μg of RNA extracted from cell lysates. The graph shows the band intensities of the scaRNAs immunoprecipitated from siRNA-treated cells relative to those from ncRNA-treated cells. The values are normalized with those of the immunoprecipitated (IPed) HEF-WDR79. Mean ± SEM, n = 3; *P < 0.05, paired t-test. (C) Immunocytochemical (WDR79) and FISH (scaRNA28) analysis of HeLa cells treated with ncRNA or siRNA for WDR79 knockdown (upper). FISH analyses of U2 snRNA and scaRNA28 are also given for the HeLa cells treated with ncRNA or siRNA for 96 h for the WDR79 knockdown (lower). In those analyses, scaRNA28 was expressed ectopically for an additional 48 h after the WDR79 knockdown and detected with Cy3-labeled scaRNA28 DNA probe (red). U2 snRNA was detected with a FITC-labeled U2 snRNA DNA probe (green). WDR79 was detected with rabbit polyclonal anti-WDR79 and FITC-conjugated anti-rabbit IgG (green). Bars indicate 5 μm. FITC and Cy3 signals were merged (Merge); DAPI for DNA staining was overlaid with the merged image. (D) HeLa cells treated for 96 h with ncRNA or WDR79 siRNA were separated into the cytoplasmic/nucleoplasmic fraction (CN) and the nucleolar fraction (No). RNAs extracted from the CN, No or total cell (To) were detected by northern blot analysis with DNA probes complementary to the RNAs indicated on the right. The extent of the WDR79 knockdown was determined by western blot analysis with antibody against WDR79 or LDHA (control).