Ecl plus kit

At POD7, after the rats were sacrificed, six 0.5 cm × 0.5 cm specimens were taken from the central tissue of the flaps in zone II of each group and kept at -80°C for immunoblotting and immunoprecipitation analysis. After the skin flap tissue was extracted, its protein content was evaluated via a BCA protein detection kit. Protein separation was carried out via PAGE (12%), followed by transferring onto the PVDF membrane. Then, nonfat milk (5%, w/v) was used for membrane blockage for 2 h at ~25°C, followed by membrane incubation with the underlying primary antibodies (for 24 h at 4°C): MMP9 (1 : 1,000), VEGF (1 : 1,000), HO1 (1 : 1,000), cadherin 5 (1 : 1,000), SOD1 (1 : 1,000), Bax (1 : 1000), eNOS (1 : 1,000), caspase 3 (CASP3) (1 : 1,000), CYC (1 : 1,000), p62 (1 : 1,000), LC3 (1 : 500), Beclin 1 (1 : 1,000), CTSD (1 : 1,000), VPS34 (1 : 1,000), and GAPDH (1 : 1,000). Then, membrane incubation was carried out with horseradish peroxidase- (HRP-) coupled IgG secondary antibody (1 : 5000) for 2 h at 25°C. The band was visually analyzed via the ECL Plus kit, and the quantification of band intensity was carried out via Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA).

The immunoblot analysis was evaluated according to a previously described method [36 (link)]. The membranes were visualized by a chemiluminescent reaction (ECL plus kit, Bio-Rad, Hercules, CA, USA) and an imaging system (ChemiDoc Touch Imaging System, Bio-Rad). The levels of target proteins were compared to those of a loading control (β-actin), and the results were expressed as a ratio of the density of each protein identified using a protein standard size marker (BIOFACT, Daejeon, Korea). The density of each band was measured using ImageJ software (version 1.49v for Windows, National Institutes of Health (NIH), Bethesda, MD, USA).

LV tissues frozen in liquid nitrogen were pulverized and homogenized in a lysis buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin (20 ml/g tissue) and 1 mM phenylmethylsulfonyl fluoride. Protein samples (20 ~ 25 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were probed with the following primary antibodies: anti-Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) antibody (Abcam, 1:1000 dilution), anti-phospho-CaMKII (Thr286) antibody (p-CaMKII, CST, 1:1000 dilution), anti-PLB antibody (Abcam, 1:1000 dilution), anti-phospho-PLB (S16) antibody (p-PLB, Abcam, 1:1500 dilution), anti-SERCA₂ antibody (Abcam, 1:1000 dilution), anti-RyR₂ antibody (Abcam, 1:1000 dilution) or anti-Na⁺/Ca²⁺-exchangers (NCX₁) antibody (Abcam, 1:1000 dilution). Appropriate secondary antibodies were used to detect the corresponding primary antibodies, and the antibody-antigen complexes in all membranes were detected by the ECL PLUS kit (Bio-Rad). The protein bands were quantified by Image Pro Plus 6.0.

Aβ₁₋₄₂ was bought from Sigma-Aldrich (St. Louis, USA) and GL Biochem. (Beijing, China) as the lyophilized powder. Hexafluoroisopropanol (HFIP) was purchased from Merck Co. (Darmstadt, Germany). Thioflavin T (ThT), thiazolyl blue tetrazolium bromide (MTT), 8-Anilinonaphthalene-1-sulfonate (ANS), and Hoechst were purchased from Sigma-Aldrich Chem. Co. (St. Louis, USA). The PC12 rat pheochromocytoma cell line was purchased from Pasture Institute (Tehran, Iran). Cell culture plates were acquired from SPL (Beijing, China). Primary antibodies (ab201060 and ab224275) and the secondary antibody (ab6721) were bought from Abcam (Abcam Inc., Cambridge, MA). ECL Plus Kit was purchased from Bio-Rad (Bio-Rad Laboratories Inc., Hercules, CA, USA). Alexa Fluor 594 (clone Poly4064) was purchased from BioLegend (San Diego, CA, USA). PVDF was purchased from GE Healthcare (Biosciences, Stockholm, Sweden). All other materials were of analytical grade.

HT22 cells were collected and total cell lysates were prepared using Phosphorylated Protein Extraction Kit (KeyGen Biotech, Nanjing, China). Equal amounts of protein extracts were mixed (5:1) with loading buffer for electrophoresis in acylamide SDS gels. After electrophoresis, samples were transferred onto polyvinylidene fluoride membrane (Millipore, MA, United States). The proteins were exposed to the specific primary antibodies against the target protein. Cross-reactivity was observed using species-specific secondary antibodies labeled with horseradish peroxidase (HRP) and ECL Plus kit in gel imaging analysis system (Biorad, CA, United States). The quantitative analysis of each bolt was carried out by Quantity One 1-D analysis software (Biorad, CA, United States).