Alliance mini hd9

Equivalent amounts of proteins associated with total cell lysates, determined by DC Protein Assay (Bio-Rad, Hercules, CA, USA), were separated on polyacrylamide gels and then transferred to PVDF (Polyvinylidene fluoride) membranes by electroblotting [29 (link)]. Blots were incubated with monoclonal rabbit anti-GCase (ab128879, Abcam, Cambridge, UK) or polyclonal rabbit anti-GAPDH (G9545, Sigma-Aldrich) primary antibodies at 4 °C overnight, followed by incubation with goat anti-rabbit HRP-conjugated (7074, Cell Signaling) secondary antibody and detection with a chemiluminescent kit (WESTAR ηC, Cyanagen, Bologna, Italy). Digital images were obtained by the chemiluminescence system Alliance Mini HD9 (UVItec, Cambridge, UK).

Total protein was extracted using RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100, 5 mM EDTA, pH 8.0) containing protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) and quantified using the Bradford method. Proteins were separated on gradient SDS-PAGE gels and transferred to nitrocellulose membranes (Whatman). Membranes were then incubated with the primary antibodies overnight at 4 °C. The following primary antibodies were used: MeCP2 (D4F3) XP Rabbit (#3456, Cell Signaling Technology); β-Actin (8H10D10) Mouse mAb (#3700, Cell Signaling Technology); PPARγ (81B8) Rabbit (#2443, Cell Signaling Technology). After incubation with the specific HRP-conjugated antibody (Vector; 1:10,000 dilution), the chemiluminescent signal was detected using Clarity and/or Clarity Max (Bio-Rad, Italy) and images were acquired with Alliance Mini HD9 (Uvitec, Cambridge, UK). Densitometric analysis was performed with ImageJ software (https://imagej.nih.gov/ij/download.html). Full and uncropped Western Blots were provided as Supplemental Material.

Cells from EBs were lysed on ice in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate) supplemented with phosphatase and protease inhibitors. Cellular lysates were used for immunoblotting with indicated antibodies. Signals in membranes were detected using ECL prime (Amersham) and images automatically captured in an Alliance Mini HD9 (UVITEC, UK) system. Acquired images were analyzed with ImageJ. Antibodies used are listed in supplementary information. For LC3B immunoblots, cells were incubated with 50 µM Chloroquine (Sigma-Aldrich, C6628) for 4 h prior lysis with ice-cold RIPA buffer. Uncropped blots can be found in Supplementary materials.

BN-PAGE gels were transferred to PVDF membranes in Dunn carbonate buffer (10 mM NaHCO₃, 3 mM Na₂CO₃) applying a constant current of 300 mA at 4 °C for 1 hr using a Mini Trans-Blot Cell (Bio-Rad). For the immunodetection of specific protein targets, blotted PVDF membranes were blocked in 5% skimmed milk in PBS-T (0.1% Tween-20) at room temperature for 1 hr and then incubated overnight with primary antibodies diluted in 3% BSA in PBS-T overnight at 4 °C. PVDF membranes were washed three times with PBS-T for 10 min, incubated with the secondary HRP- conjugated antibody for 1 hr at room temperature and washed three times with PBS-T for 10 min. Chemiluminescent signals were recorded using an Alliance Mini HD9 (UVITEC). Antibodies used are listed in the Key Resource Table. The primary antibodies against D. melanogaster UQCR-C2 and SdhA were a kind gift of Dr. Edward Owusu-Ansah (Columbia University, NY).

The expression of MT1, MT2, LC3-II, p62, BAX, and cleaved caspase 3 proteins was determined by Western blotting (n = 3). Proteins were extracted from the corneal tissues using a lysis buffer (M-PER; Pierce Biotechnology, Rockford, IL, USA) with a protease inhibitor cocktail. Lysates were centrifuged at 15,000 rpm for 10 min at 4 °C. The proteins (20 μg) in the samples were separated by 12% SDS-PAGE and were transferred to polyvinylidene difluoride membranes. The blots were then washed with TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.05% Tween-20), blocked with 5% skim milk in TBST for 1 h, and incubated overnight at room temperature with primary antibodies, including mouse anti-MT1 (catalog no. sc-390328, Santa Cruz, Dallas, TX, USA), rabbit anti-MT2 (catalog no. ab203346, Abcam, MA, USA), rabbit anti-LC3-II (catalog no. ab192890), rabbit anti-p62 (catalog no. ab109012), mouse anti-BAX (catalog no. ab216494), and rabbit anti-cleaved caspase 3 (catalog no. ab214430). After incubation with secondary antibodies, immunoreactive bands were visualized using an enhanced chemiluminescence system (ECL Blotting Analysis System; Amersham, Arlington Heights, IL, USA). The data were analyzed via densitometry (Alliance MINI HD9; UVItec Ltd., Cambridge, UK). β-Actin was used as an internal control.

Cells transduced as indicated in the text were lysed on ice in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate) supplemented with 100 mM NaF, 2 mM Na₃VO₄, 20 mM Na₄P₂0₇, as protein phosphatase inhibitors, and 1 × complete protease inhibitor cocktail (Merck). Cellular lysates were used for immunoblotting with the indicated antibodies using standard procedures. Signals in western blots were detected using ECL prime (Amersham) and images automatically captured in an Alliance Mini HD9 (UVITEC) digital imaging system equipped with a 16-bit (65,536 grey levels) scientific-grade camera with variable electronic shutter speed and 4.8 OD dynamic range. Acquired images were processed using Adobe Photoshop CS6 and analysed with ImageJ software. The antibodies used were: rabbit anti-ACC 1:1000 (Cell Signaling, 3676), rabbit anti-FASN 1:1000 (Cell Signaling, 3180), rabbit anti-G6PD 1:1000 (Cell Signaling, 12,263), rabbit anti-UCP2 1:1000 (Cell Signaling, 89,326) and mouse anti-Tubulin 1:1000 (Santa Cruz Biotechnology, sc-32293).