Extended Data Fig. 4. Properties of CMA targeted BMAL1.

a. Immunoblot for BMAL1 phosphorylated on serine 42 (pBMAL1^S42) in wild-type (WT, W) and LAMP2A knockout mice (L2A^KO, L) livers at the indicated Zeitgeber times (ZT). Total BMAL1 and actin immunoblots from Fig. 2a are shown here for comparison. Arrows: BMAL1 bands of different electrophoretic mobility. All membranes contained a common sample for normalization across membranes. b. Levels of the two bands of total (top) and pBMAL1^S42 (bottom) in WT (left) and L2A^KO mice (right) from immunoblots as in a. Values are percentage of total BMAL1 contributed by each band. n=3 mice per ZT. c. Immunoblot for total BMAL1, pBMAL1^S42 and BMAL1 acetylated on lysine 538 (AcBMAL1^K538) in mouse liver homogenates (Hom) and lysosomes (Lys) active (+) or not (−) for CMA at the time of maximal BMAL1 lysosomal degradation. LAMP2A and HSC70 from the same fractions are shown on the right. d. Immunoblot of nuclear (left) and cytosolic fractions (right) from WT and L2A^KO mice livers collected at the indicated Zeitgeber times (ZT), n=3 mice per ZT and genotype. Actin is shown as cytosolic loading control and Histone 3 as marker of the nuclear fraction. e, f. Levels of the top (left) and bottom (right) bands of BMAL1 (e) and of CLOCK (f) shown in d. Values are folds of ZT11 WT (arbitrary value of 1). n=3 mice per ZT and genotype in e and f. g. Immunoblot for total BMAL1 and pBMAL1^S42 in fractions from synchronized NIH3T3 cells cultured or not with leptomycin to block nuclear export and (+) ammonium chloride and leupeptin (N/L) to block lysosomal degradation. Right: higher exposure of lysosome lanes. h. Lysosomal degradation (fold increase upon N/L) of the top and bottom BMAL1 (top) and pBMAL1^S42 bands (bottom) in cells in g. Red dotted line: no degradation. n=3 mice per treatment. Individual values (e,f,h) and mean+s.e.m are shown. Two-way ANOVA followed by Bonferroni's multiple comparisons post-hoc test was used. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Numerical source data, statistics and exact p values and unprocessed blots are available as source data.