Ge amersham imager 600 imaging system

Tissues was homogenated with RIPA buffer which has been added with proteinase inhibitor and phosphorylated-protease inhibitor to extract protein. Protein concentration in all groups were calculated with an enhanced BCA Protein Assay Kit (P0010, Beyotime Tech, Shanghai, China) and then adjusted to same level. The same amounts of protein in each group were separated on SDS-PAGE, and then transferred to PVDF membranes. The membranes were blocked in 6% skim milk at room temperature for 2 h and then incubated at 4 °C with the relevant primary antibodies overnight. Then, membranes were incubated with the appropriate HRP-labeled secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using ECL reagent under GE Amersham Imager 600 Imaging System (GE Healthcare, Boston, MA, USA) and protein levels were digitized using ImageQuant TL software (GE Healthcare). Primary antibodies for phospho-S6 (Ser 235) and LC3B-II were purchased from Cell Signaling Technology (Beverly, MA, USA). The primary antibody for caspase-3 was purchased from Abcam (Cambridge, UK). Primary antibody for β-actin was purchased from ABClonal Biotechnology (Wuhan, China).

The carotid arteries of rats or cells were lysed with cell lysis buffer (Beyotime Institute of Biotechnology) supplemented with 0.5 mM phenylmethanesulfonylfluoride. Following centrifugation (12,000 rpm, 15 min, 4°C), protein concentrations were determined using a Bicinchoninic Acid Protein Assay Kit (Beyotime Institute of Biotechnology). The same mass of total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (0.22; Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 for 2 h at room temperature. Membrane-bound proteins were probed with the indicated primary antibody (1 : 500–1 : 1,000 dilution) overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies (1 : 5,000; Proteintech, Chicago, IL, USA) at room temperature for 1 h. Protein bands were detected with an enhanced chemiluminescence detection kit (Cell Signaling Technology) and photographed using a GE Amersham Imager 600 imaging system (GE Life Sciences, Chicago, IL, USA). Antibodies against LC3B (cat. no. 2775S), p62 (cat. no. 5114S), Beclin 1 (cat. no. 3738S), PCNA (cat. no. 13110S), and β-actin (cat. no. 4970S) were purchased from Cell Signaling Technology.

Nasal mucus (40 μl) and serum (0.5 μl) samples were resolved on 4–15% SDS-PAGE Ready Gel (Bio-Rad) under non-reducing or reducing conditions as described previously [15 (link)–17 (link)]. For western blot analysis, the gels were transferred onto PVDF membranes (Bio-Rad). Thereafter, the membranes were blocked with 8% skim milk and incubated with anti-trout IgT (rabbit pAb), anti-trout IgM (mouse monoclonal antibody (mAb)) or biotinylated anti-trout IgD (mouse mAb) antibodies followed by incubation with peroxidase-conjugated anti-rabbit, anti-mouse IgG (Invitrogen) or streptavidin (Invitrogen). Immunoreactivity was detected with an enhanced chemiluminescent reagent (Advansta) and scanned by GE Amersham Imager 600 Imaging System (GE Healthcare). The captured gel images were analysed by using ImageQuant TL software (GE Healthcare). Thereafter, the concentration of IgM, IgD and IgT were determined by plotting the obtained signal strength values on a standard curve generated for each blot using known amounts of purified trout IgM, IgD or IgT.