Western blot special reagent

Because the cells had been processed, the medium was discarded; protein lysate (Roche) was added, and total protein was separated. An aliquot of 50 g of total protein was spiked on a 12% polyacrylamide gel and subjected to electrophoresis under 100 V for 120 min. The membrane was then transformed to a PVDF film (Millipore, USA). Then, 5% powdered skim milk was closed for 1 h at room temperature, membranes were washed thrice with TBST for 10 min each time, and the closed films were incubated with primary antibody (concentration 1 : 1,000) at 4°C overnight. The membranes were then incubated with HRP-labeled anti-rabbit secondary antibody (1 : 3,000) under ambient temperature for 60 min. The membranes were washed thrice with TBST for 600 s each time. Finally, the films were imaged with western blot special reagents (Invitrogen, Carlsbad, CA, USA) for color development, and the grey values of each protein were analyzed.

The rat lung tissue homogenate, NR8383 and RLE-6TN cells were collected. The RIPA protein lysis buffer (Roche, Basel, Switzerland) was affiliated for total protein isolation. 50 µg of the total protein sampled in 12% polyacrylamide gel was electrophoresed at 100 V for 2 h and then electrically moved onto polyvinylidene fluoride (PVDF) membranes. After being sealed with 5% nonfat milk powder for an hour at regular atmospheric temperature, the membranes were flushed 3 times with TBST (10 min each) for the overnight incubation at 4°C along with the anti-SIRT1 antibody (1: 1000, Abcam, ab110304, MA, USA), anti-p-NF-κB antibody (phospho S536) (Abcam ab86299, 1: 1000), anti-NF-κB antibody (1: 1000, Abcam, ab32536, MA, USA), anti-iNOS antibody (1: 1000, Abcam, ab15323, MA, USA), anti-Bax antibody (1: 1000, Abcam, ab32503, MA, USA), anti-Bcl2 antibody (1: 1000, Abcam, ab182858, MA, USA), and anti-Caspase3 antibody (1: 1000, Abcam, ab13847, MA, USA). Following TBST washing, the membranes were incubated along with the anti-rabbit or anti-mouse secondary antibody (1: 3000) labeled by horseradish peroxidase (HRP) for 1 h [25 (link)]. TBST was applied to rinse the membranes three times (10 min each). At last, Western blot special reagent (Invitrogen) was employed for color imaging; the gray value of each protein was analyzed by Image J.