Fig 1: Diurnal Rhythms in Autophagic and Proteasomal Activity(A) Protocol for parallel time series analyses of autophagic and proteasomal flux. See STAR Methods and Figure S7A for descriptions of sample preparation.(B and C) Representative western blots of p62 and LC3b-II in the lysosome-enriched (3KP) fraction (B) and Lys48-linked polyubiquitin (K48-Ub) chains in the Cyto fraction (C). ß-actin and LDH are shown as loading controls (B and C, respectively). For (B), each lane represents 12 µL of the 3KP fraction pooled from n = 3–4 mice, representing the content obtained from 126 µg of total protein. For (C), each lane represents 19 µg of protein pooled from n = 3–4 mice. -, PBS-treated animals; L, leupeptin-treated animals; B, bortezomib-treated animals.(D–F) Quantification of circadian rhythms in autophagic and proteasomal flux in normal mouse liver using various markers (mean ± SE, n = 3–4 measurements). Data presented were concatenated from 2 independent time series experiments. For each set of data, a best fit cosine curve and rhythm parameters were generated using COSOPT and are listed in the graphs. Statistical significance was determined using one-way ANOVA.(D) Autophagic flux as measured by the increase in p62 with leupeptin in the 3KP fraction (orange squares), and proteasomal flux as measured by the increase in K48-Ub with bortezomib in the Cyto fraction (blue circles).(E) Autophagic flux as measured by the increase in p62 with leupeptin in the 3KP fraction (orange squares), and proteasomal flux as measured by the uptick in p62 content with bortezomib in the 3KP fraction (green circles).(F) Autophagic flux as measured by the increase in LC3b-II by leupeptin in the 3KP fraction (gray squares), and proteasomal flux as measured by the uptick in LC3b-II content with bortezomib in the 3KP fraction (purple circles).Also see Figures S5 and S6.
Fig 2: Circadian Characteristics of Macroautophagic Flux in Basal Mouse Liver(A) Cartoon depicting protocol for measuring macroautophagic flux.(B) Representative time series analysis of circadian rhythms in macroautophagy in basal mouse liver. Tiled images are representative western blots against p62 (top), LC3b-II (middle), and ß-actin (bottom). Standards are shown to the left, and molecular weight markers (in kilodaltons) are depicted to the right of the images. Each lane represents lysosome-enriched protein fractions isolated from 126 µg of total liver homogenate (12 µL or 6% of the total fraction). Times of tissue harvest are depicted in units of zeitgeber time (ZT), where ZT0 represents lights on (6:00 a.m. local time) and ZT12 represents lights off (6:00 p.m. local time). Collection times that occurred during subjective night are highlighted in black. -, PBS control-injected animals; +, leupeptin-injected animals.(C–E) Circadian rhythm parameter analysis of basal macroautophagic flux in mouse liver using LC3b-II (blue bars) and p62 (orange bars) as markers.(C and D) Period duration (C) and amplitude (D) expressed as a fraction of the mean (mean ± SE, n = 6).(E) Acrophase (mean ± SE, n = 6). The average phase difference ± SE between LC3b-II and p62 turnover is indicated above the graph (n = 6). The p value reflects the likelihood that the phase difference between LC3b-II and p62 acrophases is zero.(F) Western blots depicting macroautophagic flux in livers of LPS-treated mice (12 mg/kg i.p. dose given at ZT5). Each lane represents lysosome-enriched fractions isolated from 126 µg of total liver homogenate (12 µL or 6%of the total fraction) (see STAR Methods). -, PBS control-injected animals; +, leupeptin-injected animals. Data are representative of 4 independent experiments.(G and H) Quantification of macroautophagic flux using LC3b-II turnover (G) or p62 turnover (H) as a marker of macroautophagic activity (mean ± SE, n = 3 flux measurements). The p values derived by one-way ANOVA are provided in the graph.Also see Figure S1.
Fig 3: A Proteomics-Based Approach to Measuring Autophagic Flux(A) Western blots depicting p62 turnover in samples used for label-free proteomics analysis (see STAR Methods). Each lane represents 12 µL or 6% of the total fraction (see STAR Methods). Top, basal livers. Bottom, LPS (12 mg/kg given at ZT1). Below each blot, p62 turnover is calculated using either densitometry from western blotting (in units of picomoles of p62 per milligram of total protein per hour, gray values), or using FCSs obtained from proteomics (blue values for basal and red values for LPS). These data are depicted graphically in normalized form to the right of the blots. See also Figure S2A.(B) Correlation between proteomics-based and western blot-based estimates of macroautophagic flux using p62 turnover as a marker. Datapoints represent the mean of 2 turnover measurements obtained at various times of day. Blue circles, basal livers; red circles, LPS-treated mice.(C) Macroautophagy substrates identified in our proteomics time series analysis using an FDR cutoff of <5% (see STAR Methods).See also Data S1 and S2.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for Recombinant Human p62/SQSTM1 His Protein