Pan 14 3 3

After SDS-PAGE, proteins were transferred to a 0.45 mm polyvinylidene difluoride Immobilon-P membrane (ThermoFisher, IPVH00010) and blocked for 1 h at room temperature (RT) with 5% phosphate-buffered saline-Tween-20 (PBS-T) milk. Membranes were incubated overnight with primary antibodies against the following: LC3 (Novus Biologicals, NB100-2220) 1/3000; β-actin (Abcam, #ab8226) 1/10,000; Map4k3 (Cell Signaling, 9613) 1/1000; RagA (Cell Signaling, 4357) 1/1000; RagC (Cell Signaling, 5466) 1/1000; Lamtor1 (Cell Signaling, 8975) 1/1000; TFEB (Cell Signaling, 4240) 1/1000; pan 14-3-3 (Santa Cruz Biotechnology, sc-629) 1/1000; FLAG (M2) (Sigma; F1804); Raptor (24C12) (Cell Signaling, 2280) 1/1000; phospho-(Ser) 14-3-3 Binding Motif (Cell Signaling, 9601) (for detection of TFEB phosphorylation at Ser211) 1/1000; GST (Santa Cruz Biotechnology, sc-138) 1/1000, in 5% bovine serum albumin in PBS-T. Species-specific secondary antibodies were goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) or goat anti-mouse IgG-HRP (Santa Cruz, sc-2005), diluted 1/10,000 in 5% PBS-T milk and incubated for 1 h at RT. Chemiluminescent signal detection was captured with Pierce ECL Plus Western Blotting Substrate (ThermoFisher, 321-32) and autoradiography film, using standard techniques. Densitometry analysis was performed using ImageJ. All uncropped scans of immunoblots may be viewed in Supplementary Fig. 9.

Antibodies against beta-actin (1:5000, catalog no. sc-69879), Pan 14-3-3 (1:10,000, catalog no. sc-1657), and MAP4K5 (KHS, 1:500, catalog no. sc-6429) were purchased from Santa Cruz Biotechnology (Dallas, TX). α-tubulin (1:5000, catalog no. T5168) was purchased from Sigma. SGK1 (1:1000, catalog no. 3272 and 12103), pErk-T202/Y204 (1:2000, catalog no. 4370S), Erk (1:2000, catalog no. 4695S), pJNK-T183/Y185 (1:1000, catalog no. 9252), JNK (1:2000, catalog no. 9251), p-p38-T180/Y182 (1:1000, catalog no. 4511), p38 (1:2000, catalog no. 9212), pYAP-S127 (1:2000, catalog no. 4911), YAP (1:2000, catalog no. 14074), pNDRG1-T346 (1:2000, catalog no. 5482), NDRG1 (1:2000, catalog no. 9485), pS6-S235/236 (1:1000, catalog no. 2211), S6 (1:1000, catalog no. 2217), pGSK3α/β-S21/9 (1:1000, catalog no. 9331), pSrc-Y416 (1:1000, catalog no. 2123), and cleaved Caspase-3 (Asp175) (1:100 for IF, catalog no. 9664) were purchased from Cell Signaling Technology (Danvers, MA). Ki67 (1:200 for IF, catalog no. RM-9106) was purchased from Thermo Fisher. Donkey anti-rabbit Alexa Fluor® 488 (1:500, catalog no. A-21206) was purchased from Life Technologies.

BKM120 (S2247) and hydroxychloroquine (S4430) were purchased from Selleck Chemicals. BV02 (SML0140) was purchased from Sigma-Aldrich. Antibodies against the following proteins were used in this study: phospho-Akt S473 (Santa Cruz Biotechnology, sc-7985-R), Akt (Santa Cruz Biotechnology, sc-8312), α-tubulin (Santa Cruz Biotechnology, sc-8035), pan-14-3-3 (Santa Cruz Biotechnology, sc-629), 14-3-3ζ (Santa Cruz Biotechnology, sc-1019), p27 (Santa Cruz Biotechnology, sc-1641), β-actin (Santa Cruz Biotechnology, sc-47778), cleaved caspase-3 (Cell Signaling Technology, #9661), phospho-FOXO3a S253 (Cell Signaling Technology, #13129), FOXO3a (Cell Signaling Technology, #2497), LC3B (Abcam, ab48394), ATG7 (MBL, PM039), and di-methyl histone H3 (Lys9) (Cell Signaling Technology, #9753). The LC3B-EGFP expression vector (#11546) and the FHRE-luc reporter vector (#1789) were from Addgene (Cambridge, MA). The siRNAs used in the present study included ATG7 siRNA (Ambion, Silencer® Select Pre-designed siRNA, Cat#s20651), 14-3-3ζ siRNA (Invitrogen, Stealth siRNA™, cat#5480996), FOXO3a siRNA (Invitrogen, Stealth siRNA™, Cat#5436311), and negative control siRNA (Bioneer, SN-1022).

Tissues and cells were harvested and lysed in radio-immunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors. Matched protein quantities were separated by SDS–PAGE and transferred to PVDF membranes. Membranes were blocked in 3% skim milk for 2 h and then incubated with primary antibody overnight at 4 °C: Trim28 (Cell Signalling), β-actin (Santa Cruz Biotech), total HSL (Cell Signaling Technologies), pHSL-s563 (Invitrogen), total ACC (Cell Signaling Technologies), DJ-1 (Cell Signaling Technologies), pAkt-S473 (Cell Signaling Technologies), total Akt (Cell Signaling Technologies), pGSK3β-s9 (Cell Signaling Technologies), total GSK-3β (Cell Signaling Technologies), pan 14-3-3 (Santa Cruz), C/EBPα (Cell Signaling Technologies), and PPARγ (Cell Signaling Technologies). After incubation with primary antibodies, membranes were probed with their respective HRP-conjugate secondary anti mouse or anti rabbit (Biorad) antibodies in 3% skim milk for 2 h at room temperature, then visualised with chemiluminescence (Pierce). Approximated molecular weights of proteins were determined from a co-resolved molecular weight standard (BioRad, #1610374). The Image Lab Program was used to perform densitometry analyses, and all quantification results were normalized to their respective loading control or total protein.

Co-IP and Western blotting experiments were conducted in two batches. The detailed steps of co-IP were described in Method 4. The major differences between the two batches were: (1) a larger proportion of lysate was incubated with the antibody to 14-3-3 in batch 1 than that in batch 2; (2) the elution concentration in batch 1 was higher than that in batch 2, while less volume was obtained in batch 1 than in batch 2; (3) 25 µL of each input lysate or flow through sample and 15 µL eluates were loaded in batch 1 while 10 µL of each Input lysate or flow through sample and 30 µL eluates were loaded in batch 2; and (4) blocked membrane (5% milk/tris-buffered saline with Tween-20, TBST) was incubated with the primary antibody against pan-14-3-3 (Santa Cruz Biotechnology, Inc; Cat. No. sc-133233) in batch 1, while blocked membranes were incubated with the primary antibody against pan-14-3-3 (ThermoFisher Scientific, Waltham, MA, USA; Cat. No. 51-0700) and PYGM (Proteintech, Rosemont, IL, USA; Cat. No. 19716-1-AP) in batch 2 (Supplementary Figure S2). In spite of these differences between the two batches, the loading volume of IP and IgG from the same sample pool was identical, enabling comparison of protein abundance in IP vs. IgG as a validation of a specific protein binding to 14-3-3 in mouse heart.