Ecl western blotting detection kit

Protein abundance and phosphorylation were determined using a Western blot assay following standard procedures. Total proteins were lysed in RIPA lysis buffer. Isolation of cytosolic proteins was performed using the Mitochondria/cytosol Fractionation Kit (Beyotime). In brief, total protein was separated in SDS-PAGE and transferred to nitrocellulose (NC) membranes, and the following primary antibodies were used: mouse anti-MyHC1 monoclonal IgG (1:2,000, Upstate), rabbit anti-pAkt monoclonal IgG (1:1,000, Cell Signaling Technology), rabbit anti-Mul1 polyclonal IgG (1:500, Abcam), rabbit anti-pACC polyclonal IgG (1:1,000, Cell Signaling Technology), rabbit anti-pAMPKα monoclonal IgG (1:1,000, Cell Signaling Technology), rabbit anti-AMPKα monoclonal IgG (1:1,000, Cell Signaling Technology), mouse anti-Cyt C monoclonal IgG (1:1000, Abcam), and mouse anti-GAPDH monoclonal IgG (1:1,000, Goodhere). After incubation with goat anti-rabbit IgG-HRP (1:3,000, Santa Cruz Biotechnology) or goat anti-mouse IgG-HRP (1:3,000, Santa Cruz Biotechnology), immunoreactive bands were visualized with an ECL Western blotting detection kit (Millipore) using a FluorChem E System (Protein Simple Inc.). The images were analysed quantitatively using AlphaView 3.4.0 image analysis software. The results of the Western blot assay are presented as the ratio of each protein level to the corresponding control.

Brain tissue was homogenized in radioimmunoprecipitation (RIPA) lysis buffer supplemented with phenylmethylsulfonylfluoride (PMSF) solution, a protease inhibitor. The homogenates were centrifuged (12,000×g for 10 min at 4 °C), and the total protein in the supernatant was measured using the BCA Protein Assay kit (Beyotime Biotechnology). An equivalent of 80 μg protein was analyzed by 15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Roche). Membranes were blocked with 5% (w/v) bovine serum albumin (Millipore, USA) in 10 mM Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 2 h, and then probed overnight at 4 °C with specific primary antibodies: CC3 (1:500, Cell Signaling Technology) and β-actin (1:2000, Beijing Zhongshan Jinqiao Biological Technology Co. China). Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:3000, Beijing Zhongshan Jinqiao Biological Technology Co., China) for 1.5 h at room temperature. The specific protein bands were detected using enhanced chemiluminescence (ECL) Western blotting detection kit (Millipore), and the immunoreactive bands were digitally scanned and analyzed using Image J software. The band intensity of the target proteins was normalized against β-actin that was used as a loading control.

Cells and tissues were lysed in lysis buffer supplemented with proteinase inhibitors. Protein samples were separated on SDS-PAGE and transferred to the PVDF membrane (Millipore, USA). The membranes were blocked with 5% non-fat milk for 1 h and incubated with the primary LRRK2 antibody (1:10,000, rabbit, ab133474, Abcam, USA) or GAPDH antibody (rabbit, 5174T, CST, USA) at 4 °C overnight, followed by washing and incubation with HRP-conjugated goat anti-rabbit antibody (1:40,000, ab205718, Abcam, USA) for 60 min at room temperature. The ECL western blotting detection kit (WBKLS0100, Millipore, USA) was used to detect the resultant bands. All experiments were performed in triplicate at least.

HUVECs (2 × 10⁵ cells) initially seeded in 6-well plates were lysed in RIPA buffer (Thermo scientific Scientific) containing protease inhibitor cocktail (Thermo scientific Scientific) after the stimulation of LL37 and RAPA. Protein extracts were quantified and subjected to 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, United States). The membrane was blocked with 5% skimmed milk and then incubated with primary antibodies pS6 (5364, CST, 1:1000) and S6 (2217, CST, 1:1000) overnight with Tubulin (sc-5286, Santa Cruz Biotechnology, 1:1000) taken as a loading control. The membrane was incubated with horseradish peroxidase (HRP)-conjugated second antibodies for 1h and protein was visualized with ECL Western Blotting detection kit (Millipore) using Image Lab software (Bio-Rad). Quantification of band intensity was analyzed by ImageJ software.

ε-Caprolactone (ε-CL, 99%, Sigma-Aldrich) was stirred over CaH₂ for 24 h at room temperature and then it was distilled at reduced pressure under nitrogen. α-Methoxy-poly(ethylene glycol)₄₄-ω-hydroxide (MeO–PEG₄₄–OH, Mn = 2000 g/mol) was dried by co-evaporation with anhydrous toluene using a rotary evaporator. Methane sulfonic acid (MSA) was purchased from Sigma-Aldrich, and was used without further purification. Rabbit α-tubulin antibody and mouse β-Actin were purchased from Sigma-Aldrich; rabbit anti-CD31 (GB11063-2) was purchased from Servicebio; rabbit anti-Bcl-2 antibody and rabbit anti-Ki67 were purchased from Bioss; rabbit anti-MDR1/ABCB1 (E1Y7B) antibody was purchased from Cell Signaling Technology; horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell signaling Technology. LysoTracker^® Red DND-99 and DAPI were obtained from Thermo Scientific. Reactive oxygen species (ROS) detection kit and Annexin V-FITC apoptosis detection kit were purchased from Biovision. ECL western blotting detection kit was purchased from Millipore.

The cellular lysates were prepared by suspending 5×10⁵ cells in 100 μL of lysis buffer (137 mM NaCl, 15 mM EGTA, 0.1 mM sodium orthovanadate, 15 mM MgCl₂, 0.1% Triton X-100, 25 mM MOPS, 100 μM phenylmethlsulfonyl fluoride, and 20 μM leupeptin, pH 7.2). The cells were disrupted by sonication and extracted at 4°C for 30 min. The total protein in the lysates was quantified using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). The proteins were electrotransferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). Detection of specific proteins in the lysates was performed using an ECL Western Blotting Detection Kit (Millipore) according to the manufacturer's instructions.