P0044

HeyA8 and ES-2 cells were processed for nuclear and cytoplasmic extracts. Cells were scraped, resuspended in hypotonic lysis buffer 10mM HEPES pH7.9, 10mM KCl, 0.1mM EDTA, protease inhibitors (P8849, Sigma-Aldrich) and phosphatase inhibitor cocktails 2 and 3 (P0044, P5726, Sigma-Aldrich) and incubated on ice for 20 min. After the addition of 0.25% Igepal-630 (NP40) (Sigma-Aldrich), samples were centrifuged at 3000 rpm for 5 min and supernatants containing the cytoplasmic extracts were recovered. Nuclear pellets were resuspended in 20mM HEPES pH7.9, 0.4M NaCl, 1mM EDTA with protease and phosphatase inhibitors. After three cycles of vortex and ice, samples were centrifuged at 12000 rpm for 20 min and the supernatants containing the nuclear extracts were collected.
Proteins were separated on 4–12% NuPAGE Novex Bis-Tris Protein gradient polyacrylamide gels (Thermo Fisher Scientific) and blotted onto nitrocellulose. Membranes were quenched with 5% blotto (BioRad) [42 (link)] and the proteins detected with: rabbit anti-ZNF521 (EHZF S15 sc-84808, Santa Cruz Biotechnology, DBA, Milan, Italy) at 1:1000, rabbit anti- HDAC1 (H3284, Sigma) 1:10000. Secondary rabbit HRP antibody at 1:2000 (65–6120 Thermo Fisher Scientific) was detected by the ImmunoCruz Western blotting luminal reagent (sc-2004, Santa Cruz, Biotechnology) and exposure to autoradiographic film (GE Healthcare, Milan, Italy).

Cells were lysed in RIPA buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 7.4, 150 mM NaCl, 0.5 mM EDTA) or in buffer for immunoprecipitation (IP buffer: 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 10% glycerol), supplemented with protease inhibitor cocktail (6 μg/ml chymostatin, 0.5 μg/ml leupeptin, 10 μg/ml antipain, 2 μg/ml aprotinin, 0.7 μg/ml pepstatin A and 10 μg/ml 4-amidinophenylmethanesulfonyl fluoride hydrochloride; Sigma-Aldrich) and phosphatase inhibitor cocktails (P0044 and P5726, Sigma-Aldrich). Protein concentration was assessed with BCA Protein Assay Kit (Thermo Fisher Scientific). Then, 25–30 μg of total protein/sample were resolved on 10–14% polyacrylamide gels, transferred to nitrocellulose membrane (Whatman), that was incubated with specific primary and secondary antibodies. For signal detection either ChemiDoc imaging system (Bio-Rad) or Odyssey infrared imaging system (LI-COR Biosciences) were employed. Densitometry analysis of protein bands was performed using ImageJ Software [43 (link)].

Animals used for protein analysis were i.p anaesthetized (as described above) and perfused for 1 min with cold 0.1 M PBS (pH 7.4). Subsequently, the entire ipsilateral hippocampus was dissected out, snap frozen individually in liquid nitrogen, and stored at − 80 °C. Total protein was extracted by solubilization of samples on lysis buffer, containing 25 mM HEPES, 2% Igepal, 5 mM MgCl₂, 1.3 mM EDTA, 1 mM EGTA, 0.1 M PMSF, and protease (1:100, P8340, Sigma Aldrich) and phosphatase inhibitor cocktails (1:100, P0044, Sigma Aldrich), for 2 h at 4 °C. After solubilization, samples were centrifuged at 13,000 rpm for 5 min at 4 °C and the supernatants collected. The hippocampus from each animal was analyzed separately. Total protein concentration was determined with a commercial Pierce® BCA Protein Assay kit (#23225, Thermo Scientific) according to manufacturer’s protocol. Protein lysates were stored at − 80 °C until used for the protein microarray analysis.

Enriched mitochondrial fractions were obtained from cortex and cerebellum of 4-month old and 2-y mice by mechanical cell disruption using a glass-Teflon homogenizer and subsequent centrifugation as previously described^{77 (link)}. The enrichment of mitochondria in the mitochondria fraction was in the range of 30 to ~300-fold as judged by the ratios of mitochondrial over cytosolic markers (Supplementary Table S1). The mitochondria-enriched fraction was resuspended in hypo-osmotic 20 mM HEPES buffer, pH 7.4, supplemented with phosphatase inhibitors (Sigma #P0044) and proteolytic inhibitors (Sigma #P2714), and stored at −80 °C. A fraction of the mitochondria-enriched samples were further centrifuged at 16,000 g for 10 min and pellet (mitochondria membrane fractions) was resuspended in RIPA buffer for the quantification of membrane-bound palmitoyl-Drp1 levels (see below). Protein concentration was evaluated using a BCA Protein assay kit (Pierce) following manufacturer’s recommendation. Absorbance was recorded at 560 nm with the use of a Tecan Infinite M200 plate reader (Tecan, Austria). Other details were included in Supplementary Methods.

For in vitro analysis, GST-MoRgs7, GST-MoRgs7^5A, and His-MoSep1 were expressed in E. coli DE3 cells and purified [41 (link)]. We used the Pro-Q Diamond Phosphorylation gel stain (Thermo Fisher Scientific), a phosphor-protein gel-staining fluorescence dye in this assay. A kinase reaction buffer (100 mM phosphate-buffered saline, pH 7.5, 10 mM MgCl₂, 1 mM ascorbic acid) was mixed with MoRgs7 and His-MoSep1, MoRgs7^5A, and His-MoSep1, respectively. The subsequent experiments were performed according to the previously described protocol [21 (link)].
For in vivo analysis, conidia were prepared from various transformants as described above and were filtered through three layers of lens paper before resuspending in sterile water (2 ×10⁵ spores mL^-1) [42 (link)]. For appressorium protein extraction, droplets (5 mL) of spore suspensions were placed on strips of onion epidermis, incubated under humid conditions at room temperature for 6 h, and onion epidermis grounded for protein extraction [43 (link)]. Protein extraction was the same as described above and phosphorylation analysis was performed as according to the protocol, phosphatase inhibitors (P0044, sigma) and alkaline phosphatase (P6774, sigma) [21 (link)].