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. 2023 Jun 22;299(8):104948. doi: 10.1016/j.jbc.2023.104948

Figure 3.

Figure 3

Comparison of proteasome assembly and activity in C1 and WT cells.A, native gel electrophoresis of lysates from C1 or WT HCT116 cells immunoprobed for CP subunit β5. Whole cell extracts (∼30 μg) from WT or C1 cells were subjected to native gel electrophoresis and then incubated in solubilization buffer before transferring onto a PVDF membrane and immunoprobing with anti-β5 antibodies (left). Quantification of the C1:WT ratio of RP-CP-RP, RP-CP, and CP within the corresponding boxed regions (right). B, in-gel peptidase assay by using native gel electrophoresis with Suc-LLVY-AMC for lysates from WT and C1 cells. Equivalent quantities of WT and C1 cellular lysates were separated by native gel electrophoresis prior to staining with Suc-LLVY-AMC peptide (left). Quantification of the highlighted regions representing RP-CP-RP, RP-CP, and CP bands of C1 was performed by normalizing with the respective bands for WT and plotted as a bar graph (right). C, immunoprobing (left) and quantification (right, n = 3) of whole cell extracts from parental (WT) and C1 cells for K48-linked ubiquitin chains with β-actin as a loading control and used for normalization. n.s., not significant (p = 0.37, two-tailed paired t test). CP, core particle; PVDF, polyvinylidene fluoride; RP, regulatory particle.