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. 2021 Feb 22;49(5):2740–2758. doi: 10.1093/nar/gkab081

Figure 5.

Figure 5.

Hsp70 regulates CHIP-mediated p63 ubiquitination. (A) CHIP promotes TAp63 ubiquitination. H1299 cells were transfected with plasmids expressing HA-ubiquitin (Ub), T7-TAp63, Myc-Itch, Flag-CHIP, or Flag-CHIPΔU, as indicated. Cell extracts were immunoprecipitated with a T7-specific (69522-4) antibody against TAp63, and analyzed by immunoblotting with antibodies to HA (12CA5). Direct western blots for TAp63 (T7), Itch (Myc) and CHIP (Flag, M2) are shown in the bottom panels. (B) Similar to (A), except that the T7-ΔNp63 construct was used. (C) H1299 cells were transfected with Hsp70-siRNA-2 or control-siRNA constructs. 40 h later, the cells were further transfected with a plasmid expressing HA-Ub. Lysates were immunoprecipitated with a TAp63-specific (Poly 618902) antibody and analyzed by western blotting with an HA-specific (12CA5) antibody to detect ubiquitinated TAp63. Western blots for TAp63, Hsp70, CHIP, and actin are shown in the lower panel. (D) Similar to (C), except that SCC9 cells and an ΔNp63-specific (Poly 619002) antibody were used. (E) H1299 cells were transfected with plasmids expressing CHIP, HA-Ub, or empty vector. Thirty hours later, the cells were treated with Ver-155008 (25 μM), an inhibitor of Hsp70, and immunoprecipitated with a TAp63-specific (Poly 618902) antibody and analyzed by western blotting with an HA-specific (12CA5) antibody to detect ubiquitinated TAp63. The western blots for TAp63, Hsp70, CHIP and actin are shown in the lower panel. (F) Similar to (E), except that SCC9 cells and an ΔNp63-specific (Poly 619002) antibody were used. (G) CHIP promotes TAp63 and ΔNp63 ubiquitination in vitro. Western blot of the in vitro ubiquitination reaction probed with a Ub-specific antibody (BD, Biosciences) to detect ubiquitinated p63. Direct Western blots for CHIP (anti-GST) and p63 (anti-His) are shown in the lower panel. (H) Western blot analysis of a coupled in vitro ubiquitination-immunoprecipitation. After the in vitro ubiquitination, the samples were immunoprecipitated with a TAp63-specific (Poly 618902) antibody and analyzed by western blotting with a ubiquitin-specific antibody (550944, upper image). Direct western blots for TAp63 (Poly 618902), CHIP (H-231), and Hsp70 (C92F3A5) are shown in the lower panels. (I) Similar to (H), except that the reaction was immunoprecipitated with an ΔNp63-specific (Poly 619002) antibody as indicated. All experiments were performed in triplicate.