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

Figure 3.

Figure 3.

CHIP decreases the levels of TAp63 and ΔNp63. (A) The expression of CHIP decreases the levels of TAp63 in cells. Plasmids expressing T7-TAp63, or in combination with MDM2, Myc-Itch, or Flag-CHIP, were transfected into H1299 cells and analyzed by immunoblotting with T7 tag-specific (69522-4), MDM2-specific (SMP14), Myc tag-specific (9E10), Flag tag-specific (M2) antibodies. (B) H1299 cells were transfected with T7-TAp63 or T7-ΔNp63, or together with Flag-CHIP, and analyzed by immunoblotting. (C) H1299 cells were transfected with increasing amounts of the Flag-CHIP expression plasmid. The levels of expression of Flag-CHIP and endogenous TAp63 protein were determined by immunoblotting with Flag-specific (M2) and TAp63-specific (Poly 618902) antibodies. An antibody against actin was used as a loading control. (D) Similar to the (C), except that SCC9 cells were transfected. The protein levels of endogenous ΔNp63 and CHIP expression were detected by western blotting with Flag-specific (M2) for CHIP and ΔNp63-specific (Poly 619002) antibodies. (E) H1299 cells were transfected with CHIP-siRNA-1 or CHIP-siRNA-4 and analyzed by immunoblotting with CHIP-specific (H-231) and TAp63-specific (Poly 618902) antibodies. (F) Similar to (E), except that SCC9 cells were used. The protein levels of endogenous ΔNp63 and CHIP were detected by western blotting with CHIP-specific (H-231) and ΔNp63-specific (Poly 619002) antibodies. (G) Cell extracts were prepared from Chip−/−and Chip+/+ MEFs. The amount of endogenous Chip, TAp63, and ΔNp63 proteins were detected by western blotting with Chip-specific (EPR4447), TAp63-specific (938102), ΔNp63-specific (N-16) antibodies. (H) H1299 cells were transfected with CHIP, or CHIP mutants (K30A or H260Q), or empty vector, and analyzed by western blotting with TAp63-specific (Poly 618902), CHIP-specific (H-231) antibodies. (I) Similar to (H), except that SCC9 cells and ΔNp63-specific (Poly 619002) antibody were used. All experiments were performed in triplicate.