Nuclear Proteins

After being extracted from the frozen pulverized left ventricles or cultured cells and quantified, total proteins were prepared for western blot analysis and normalized to the matched total proteins or GAPDH according to our previous study^{52 (link)}. Briefly, separated proteins were incubated with the indicated primary antibodies overnight at 4 °C and with secondary antibodies for 60 min at room temperature. Nuclear and cytosolic protein fractions were separated using a commercial kit (Thermo Fisher Scientific) according to the manufacturer′s protocol. Proteins from cytosolic lysates were normalized to GAPDH, whereas proteins from nuclear lysates were normalized to PCNA.
Isolated total mRNA from hearts and cultured cells was reversely transcribed to complementary DNA (cDNA) with Transcriptor First Strand cDNA Synthesis Kit (Roche (Basel, Switzerland), 04896866001). Transcriptional level of target genes were normalized to Gapdh, and the primers for quantitative real-time PCR are shown in Supplementary Table 3.

The coding sequences of AtSCC3, AtSYN4, AtBMI1A, AtBMI1C, and AtBMI1B were subcloned into EcoRI/SalI-treated vector pCambia1300-35S-N1-FLAG, respectively. Primers used were shown in Supplementary Data 8. The plasmids containing AtSCC3-FLAG, AtSYN4-FLAG, AtBMI1A-YFP, AtBMI1B-YFP, or AtBMI1C-YFP expressing cassettes were introduced into Agrobacterium tumefaciens GV3101 by electroporation and then incubated in LB (with 50 mg/L kanamycin and 25 mg/L gentamycin) plate medium at 30 °C for 48–72 h to OD₆₀₀ = 0.8. For co-expression of AtBMI1A/B/C-YFP and AtSYN4-FLAG with or without AtSCC3-FLAG, the corresponding Agrobacterium tumefaciens strains were mixed equally and then co-infiltrated into tobacco (N. benthamiana) leaves using an injection syringe. 48 h later, the leaves were collected (about 2 g of infiltrated leaves) and ground into good powder in liquid nitrogen for nuclear protein extraction. Samples were re-suspended in 25 mL CLB1 (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.04% (v/v) β-mercaptoethanol, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1×Cocktail) and incubated at 4 °C for 30 min. The suspensions were first filtrated through a monolayer layer of Miracloth (Millipore) and then filtrated through a double layer of Miracloth, and centrifuged at 3000×g for 20 min at 4 °C. The pellets were washed twice with 1 mL CLB2 (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 ×Cocktail) and re-suspended in 500 µL CLB2 added with 25 µL 5% (w/v) SDS. The suspensions were sonicated five times and centrifuged at 12,000×g for 10 min at 4 °C. Another 1.5 mL CLB2 buffer was added for resuspension. The nuclear proteins were incubated with 50 µL Anti-GFP mAb-Magnetic Beads (MBL) and incubated overnight with rotation at 4 °C. The beads were washed 5 times with washing buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) TritonX-100, 1 mM EDTA, 1 × Cocktail). The proteins were released by boiling at 100 °C for 10 min and subjected to Western blotting assays using anti-GFP (Abiocode; M0802-3a, 1:1000 dilution) and anti-FLAG (Sigma; F1804, 1:3000 dilution) antibodies. Blotting signals were detected by the PMCapture software (Version 1.00) of a Chemiluminescence Imaging System (Tanon 5500, Shanghai, China). Proteins were detected by western blots with anti-GFP or anti-FLAG antibodies. Values came from three biological replicates.

hDF cells were collected and washed with cold PBS (2.7 mM KCl, 1.2 mM KH₂PO₄, 8.1 mM Na₂HPO⁴, 138 mM NaCl [pH 7.4]). Total proteins were solubilized in lysis buffer (20 mM Tris–HCl [pH 7.4], 1 mM ethylenediaminetetraacetic acid (EDTA), 0.4 mM NaF, 0.04 mM Na₃VO, 1% NP40, protease inhibitors) and sonicated in ice. Extraction of separate cytoplasmic and nuclear protein fractions was performed with NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated by SDS-PAGE and transferred on PVDF membrane (Millipore, Bedford, Massachusetts, USA). The level of expression of TMEM175, LC3I/II, p62, LAMP1, TFEB, GAPDH, and PARP1 was determined by immunoblot analysis using anti-LAMP1 (ab25630, Abcam, Cambridge, UK, 1:1000), anti-LC3B (2775, Cell Signaling, Danvers, Massachusetts, USA, 1:1000/), anti-TMEM175 (19,925–1-AP, Proteintech, Manchester, UK, 1:1000) and anti-p62/SQSTM1 (P0067, Sigma-Aldrich, St. Louis, MO, USA, 1:15,000), anti-TFEB (A303-673A, Bethyl Laboratories (Waltham, MA, USA), 1:1000), anti-PARP1 (66,520-1Ig, Proteintech, Manchester, UK, 1:20,000), and anti-GAPDH (sc-32233, Santa Cruz Biotechnology (Dallas, Texas USA), 1:1000). Cytoplasmic proteins were normalized to GAPDH, whereas nuclear proteins were normalized to PARP1.
Immunoprecipitation (1 mg total lysate) was performed using the anti-Akt antibodies (Immunological Sciences, Cat. N. MAB-94320) complexed to protein G-Sepharose (Invitrogen, Cat. N. 101,243). The immunoprecipitated proteins were resolved on 10% SDS–PAGE, transferred for 2 h at room temperature on PVDF membrane, and detected by immunoblotting with GFP (1:1000) (Synaptic System, Cat. N. 132 002) and Akt (1:1000). HRP-conjugated secondary antibodies (Immunological Sciences, Cat. N. IS20402) were used at 1:5000 dilution. Protein bands were detected by ECL and visualized by Quantity One software (Bio-Rad Laboratories).
The mean standard deviations of 3 independent experiments were analyzed as multiple datasets with ANOVA test for multiple comparisons or with Student’s t-test. Data were plotted as histogram representation. A value of p < 0.05 was considered statistically significant.