Fig 1: EBV-BPLF1, HSV-1-UL36, HCMV-UL48 and KSHV-ORF64 interact with SQSTM1/p62 but differentially affect its ubiquitination.(A) HeLa cells were transfected with FLAG-tagged BPLF1, UL36, UL48 or ORF64 and cell lysates were immunoprecipitated with anti-FLAG coated beads. Comparable levels of co-immunoprecipitated SQSTM1/p62 were detected in all samples. Western blots from one representative experiment out of two where the viral proteins were tested in parallel are shown. (B,D) BPLF1, UL36, UL48 or ORF64 were co-transfected in HeLa cells together with HA-UbK48 or HA-UbK63 and endogenous SQSTM1/p62 was immunoprecipitated with specific antibody in denaturing conditions. Polyubiquitin chains were detected by probing western blots with antibodies to the HA tag. Western blots from one representative experiment out of two are shown. (C,E) The intensity of the ubiquitin smears was quantified by densitometry in two independent experiments. The Mean ± SD of smear intensity in test samples relative to the vector control after normalization to the intensity of the immunoprecipitated SQSTM1/p62 bands are shown. The viral enzymes removed K48-linked polyubiquitin chains from SQSTM1/p62 with comparable efficiency (B,C), whereas UL36 exhibited significantly weaker activity against K63-linked chains (D,E).
Fig 2: The inhibitory effects of MT on autophagy reduces oxidative damage in mouse follicular GCs. Mice were injected i.p. with melatonin (15 mg/kg) or 0.5% ethanol saline once daily at 8:00 a.m. for 7 days. From day 3 to day 7, mice received an additional i.p. injection of 3-NP (50 mg/kg) or 0.5% ethanol saline at 8:00 p.m. each day. Ovaries were collected 24 h after the final injection. Immunohistochemical staining of GCs in ovarian sections was detected using anti-MAP1LC3B (A) and anti-SQSTM1 (B). Bar, 100 µm. O, oocyte; GC, granulosa cells; B, basement membrane; T, theca cells. Areas outlined in red are enlarged in lower panels. (C) Immunoblotting analysis of MAP1LC3B and SQSTM1 in ovarian GCs harvested from mice subjected to the indicated treatments. (D–F) Quantification of total MAP1LC3B expression, MAP1LC3B-II accumulation, and SQSTM1 degradation. TUBA1A served as the control for loading. Data represent mean ± S.E; n = 3. **P < 0.01; NS, not significant, P > 0.05. (G) Viability of ovarian GCs retrieved from mice with indicated administration was assessed using CCK-8 assay. Data represent mean ± S.E; n = 3 in each group. **P < 0.01; NS, not significant, P > 0.05 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
Fig 3: L50A/L59A mutations in UXT specifically disrupt interaction between p62 and UXT.a Interactions between p62 and UXT(L50P/L59P), represented as L/P (upper panel) and p62 and UXT(L50A/L59A), represented as L/A (lower panel) were analyzed. b HeLa/p62KO cells were co-transfected with oligomerization-defective p62(ΔPB1) and either UXT wild type, UXT(L50A/L59A), or UXT(L50P/L59P) mutants, and were stained with anti-p62 antibody followed by anti-mouse IgG-Alexa-350. Representative images are shown. p62(ΔPB1) clusters induced by UXT are indicated with white arrows. Scale bar, 10 μm. c Clustering indexes of p62 (left) and UXT (right) were analyzed under each condition, and are shown as scatter plots (n = 98, 82, and 22 cells examined over three independent experiment, mean ± SEM). ***p < 0.001; ns not significant (two-sided Mann–Whitney test). d The effects of L50P/L59P and L50A/L59A UXT mutations on the amount of detergent-insoluble SOD1(A4V) aggregates. e The degree of HA-UXT mutants detected in the detergent-insoluble faction was normalized against that of HA-UXT wild type and shown as bar graphs (n = 3, mean ± SEM). ***p < 0.001; ns not significant (one-way ANOVA using Bonferroni’s multiple comparison test). f SEC profiles of GFP-UXT(L50A/L59A) and GFP-UXT(L50P/L59P) as detected by GFP fluorescence in the eluant.
Fig 4: Hsa_circ_0000199-miR-613/miR-206 axis participated in regulating autophagy of triple-negative breast cancer (TNBC) cells. (A) Beclin-1, LC3-II, p62 and Atg5 expressions were determined in MDA-MB-231 and MDA-MB-468 cell lines treated by NC, si-NC, si-hsa_circ_0000199 and si-hsa_circ_0000199+miR-206 inhibitor. **: P<0.01; ***: P<0.001. (B) Beclin-1, LC3-II, p62 and Atg5 expressions were detected among MDA-MB-231 and MDA-MB-468 cell lines of NC, si-NC, si-hsa_circ_0000199 and si-hsa_circ_0000199+miR-613 inhibitor groups. **: P<0.01; ***: P<0.001. (C) Monodansylcadaverine (MDC) fluorescence intensity was monitored among MDA-MB-231 and MDA-MB-468 cell lines transfected by NC, si-NC, si-hsa_circ_0000199 and si-hsa_circ_0000199+miR-613 inhibitor. (D) MDC fluorescence intensity of MDA-MB-231 and MDA-MB-468 cell lines was determined among NC, si-NC, si-hsa_circ_0000199 and si-hsa_circ_0000199+miR-206 inhibitor groups.
Fig 5: The effects of calcitriol (0.5 µg/kg) on Nrf2 signaling after TBI. a Representative images of Western blot staining for Keap1, cytoplasmic Nrf2, and nuclear Nrf2. b Statistical graphs of Keap1 protein expression and Nrf2 translocation. c, d Representative immunofluorescence images and statistical graphs of p62 and Keap1 co-expression (Bar = 50 µm). e, f Representative immunofluorescence images and statistical graphs of Nrf2 translocation (Bar = 50 µm). g The relative mRNA expression level of Nqo1. h The relative mRNA expression level of Gclc. i The relative mRNA expression level of HO1. Data are presented as means ± SEM (n = 5). *P < 0.05 and **P < 0.01 versus the indicated groups. (Sham: sham-operated group; TBI: TBI model group; Calcitriol: TBI + calcitriol treatment group)
Supplier Page from Abcam for Anti-SQSTM1 / p62 antibody