Journal: bioRxiv

Article Title: UFD1 Recognition of Initiator and Proximal Ubiquitin Drives p97-Mediated Substrate Unfolding enhanced by FAF1, FAF2, and UBXD7

doi: 10.64898/2026.03.13.711538

Figure Lengend Snippet: a, FACS-based analysis of the pexophagy flux. Representative FACS data (mKeima-SKL 561/488 ratio) for WT, FAF2 KO, or FAF2 KO stably expressing the indicated 3HA-FAF2 mutants, with the percentage of pexophagy-positive cells indicated. b, Quantitative analysis of the pexophagy flux for cells from a. Data represent the mean percentage of pexophagy-positive cells from three independent experiments. Error bars represent mean ±SEM. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001; ns. not significant. c , Cycloheximide (CHX) chase assay in HEK293T cells transfected with the indicated siRNAs for 72 h and treated with CHX for 0–6 h. Proteins were detected by western blotting using the indicated antibodies. d, CHX chase assay in HEK293T cells depleted of FAF1 and UBXD7 by siRNA (72 h) and rescued with HA-tagged WT, QY/AA (Q511A/Y515A), or ΔUBX mutant FAF1 (48 h). Cells were treated with CHX for 0–3 h. Proteins were analyzed by western blotting using the indicated antibodies. Graphs on the right show quantification of protein levels normalized to the signal at 0 h, defined as 100% (p21 or TXNIP; n = 2 biological replicates).

Article Snippet: The following antibodies were used: p21 (Cell Signaling Technologies, #2947, 1:1000), p27 (Cell Signaling Technologies, #3686, 1:1000), TXNIP (Cell Signaling Technologies, #14715, 1:1000), HIF-1α (Cell Signaling Technologies, #14179, 1:1000), FAF1 (Cell Signaling Technologies, #4932, 1:1000), UBXD7 (Thermo Fisher Scientific, #PA5-71280, 1:500), HA (Cell Signaling Technologies, #3724, 1:1000), and CDT1 (Cell Signaling Technologies, #8064, 1:1000).

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Mutagenesis