Figure 2.
Expression and incorporation of V5/HIS-tagged PSMB10 single variants into proteasome complexes in HEK293T cells. (A) HEK293T cells were treated with IFN-γ (200 U/ml) for 24 h, followed by transfection with various C-terminally V5/HIS-tagged PSMB10 constructs. Proteins were extracted and analyzed by SDS-PAGE and western blotting using antibodies specific for V5, UBE2L6, and β-actin (loading control), as indicated. For V5 immunostaining, both short and long exposure times are shown. The immature (32.627 kDa) and mature (28.339 kDa) forms of PSMB10 protein (β2i) are marked with arrows, with the mature form visible only under long exposure. Representative data from one experiment are shown. The right panel displays densitometric analyses of V5 immunoreactive bands. Data are presented as fold-change mean values ± SEM for each variant relative to their wild-type counterpart (set to 1, indicated by the gridline) from three independent experiments. Statistical significance was assessed using a paired t test (*P < 0.05, **P < 0.01). (B) Protein samples described in A were analyzed by native-PAGE, followed by western blotting using a V5-specific antibody. Arrows indicate the positions of the 30S, 26S, 20S, and assembly intermediate proteasome complexes. The right panel shows densitometric analyses of V5 immunoreactive bands. Data are presented as band intensity values ± SEM from three independent experiments. Statistical significance was assessed using a paired t test (*P < 0.05, **P < 0.01). (C) HEK293T cells were treated with IFN-γ (200 U/ml) for 24 h and subsequently transfected with various C-terminally V5/HIS-tagged PSMB10 constructs. Protein lysates were extracted and analyzed by SDS-PAGE, followed by western blotting using antibodies specific for the immunoproteasome subunits PSMB8, PSMB9, and PSMB10, as indicated. β-actin was used as a loading control to ensure equal protein loading. The right panel presents densitometric analyses of PSMB8, PSMB9, and PSMB10 immunoreactive bands, with data expressed as fold-change mean values ± SEM for each variant relative to the PSMB10 (WT)-V5/HIS condition (normalized to 1, as indicated by the gridline) across three independent experiments. Statistical significance was determined using a paired t test (*P < 0.05, **P < 0.01). WT, wild-type. Source data are available for this figure: SourceData F2.

Expression and incorporation of V5/HIS-tagged PSMB10 single variants into proteasome complexes in HEK293T cells. (A) HEK293T cells were treated with IFN-γ (200 U/ml) for 24 h, followed by transfection with various C-terminally V5/HIS-tagged PSMB10 constructs. Proteins were extracted and analyzed by SDS-PAGE and western blotting using antibodies specific for V5, UBE2L6, and β-actin (loading control), as indicated. For V5 immunostaining, both short and long exposure times are shown. The immature (32.627 kDa) and mature (28.339 kDa) forms of PSMB10 protein (β2i) are marked with arrows, with the mature form visible only under long exposure. Representative data from one experiment are shown. The right panel displays densitometric analyses of V5 immunoreactive bands. Data are presented as fold-change mean values ± SEM for each variant relative to their wild-type counterpart (set to 1, indicated by the gridline) from three independent experiments. Statistical significance was assessed using a paired t test (*P < 0.05, **P < 0.01). (B) Protein samples described in A were analyzed by native-PAGE, followed by western blotting using a V5-specific antibody. Arrows indicate the positions of the 30S, 26S, 20S, and assembly intermediate proteasome complexes. The right panel shows densitometric analyses of V5 immunoreactive bands. Data are presented as band intensity values ± SEM from three independent experiments. Statistical significance was assessed using a paired t test (*P < 0.05, **P < 0.01). (C) HEK293T cells were treated with IFN-γ (200 U/ml) for 24 h and subsequently transfected with various C-terminally V5/HIS-tagged PSMB10 constructs. Protein lysates were extracted and analyzed by SDS-PAGE, followed by western blotting using antibodies specific for the immunoproteasome subunits PSMB8, PSMB9, and PSMB10, as indicated. β-actin was used as a loading control to ensure equal protein loading. The right panel presents densitometric analyses of PSMB8, PSMB9, and PSMB10 immunoreactive bands, with data expressed as fold-change mean values ± SEM for each variant relative to the PSMB10 (WT)-V5/HIS condition (normalized to 1, as indicated by the gridline) across three independent experiments. Statistical significance was determined using a paired t test (*P < 0.05, **P < 0.01). WT, wild-type. Source data are available for this figure: SourceData F2.

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