Horseradish peroxidase linked secondary antibody

The Hrd1 protein level was determined by using Western blot analysis as previously described.21 (link) Briefly, the tissues or cells were dissociated on ice and homogenized in a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) containing 50 μL/mL protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Beyotime). The protein concentration in the supernatants was determined by the BCA method. Samples containing 20 µg of protein were boiled and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% Tris-glycine gels and electrophoretically transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% fat-free milk in Tris-buffered solution containing 0.05% Tween-20 for 1 hour at room temperature and then incubated with rabbit anti-human Hrd1 (1:5,000; Santa Cruz Biotechnology) overnight at 4°C. The membrane was washed and incubated with a horseradish peroxidase-linked secondary antibody (1:2,000, Beyotime) and finally processed by using an ECL chemiluminescence reaction kit (Millipore, Billerica, MA, USA), followed by exposure to medical film. The band density of the target protein relative to that of β-actin was quantified with Bio-Rad Quantity One 1-D Analysis Software (Bio-Rad Laboratories, Hercules, CA, USA).

At 24 hours after the TBI challenge, the lung samples were homogenized in protein extract solution and protease inhibitors (Beyotime Biotechnology, Shanghai, China) and centrifuged at 12 000 rpm for 10 minutes at 4°C. The total protein concentration was evaluated by the BCA protein assay kit (Thermo Scientific, IL, USA). Equal amounts of total protein were loaded per well on a 12% sodium dodecyl sulfate-polyacrylamide gel and transferred into a polyvinylidene difluoride membrane. The membranes were blocked with 5% BSA for 2 hours at room temperature. Then the membranes were incubated with appropriate dilution of specific primary antibodies (anti-RAGE 1: 1000 and anti-GAPDH 1: 1000) for 12 hours at 4°C. Then the membranes were incubated with the horseradish peroxidase-linked secondary antibody (1: 1000; Beyotime Biotechnology, Shanghai, China). Protein bands were demonstrated by enhanced chemiluminescence Western blot kit (Thermo Scientific, IL, USA). Signals were densitometrically assessed and normalized to GAPDH signals.

Primary fibroblasts from PC4^+/+ and WT mice were collected, washed and lysed with a lysis buffer (Cell Signaling Technology, USA) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific Inc.) for 0.5 hours on ice. Total protein was quantitated with a bicinchoninic acid assay kit (Thermo Fisher Scientific Inc.). Each sample was denatured with a loading buffer (Beyotime, China), separated by electrophoresis on a 12% gel and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked with an animal-free blocking solution (Cell Signaling Technology) and incubated with PC4 primary antibody (11 000, ab84459, Abcam) for 16 hours at 4°C. The membranes were washed thrice with Tris-Buffered Saline Tween-20 (TBST) (5 minutes each wash) and incubated for 1–2 hours with horseradish peroxidase-linked secondary antibody (Beyotime) at 25°C. The intensity of bands was visualized and determined using an enhanced chemiluminescence detection system (Bio-Rad Laboratories). Images were quantified with ImageJ software. Actin was used as a loading control. PC4/actin shows relative expression level of PC4 protein.

Cell cultures were harvested and homogenized in ice-cold radioimmunoprecipitation assay buffer with protease inhibitors (Beyotime, Shanghai, China) and incubated for 30 mins at 4℃. After centrifugation, the supernatants, which contained our target proteins, were transferred to a new vial for storage at −70℃ or immediate use. A BCA protein assay kit was then used to quantify the concentrations of proteins (Beyotime, Shanghai, China). The proteins (40 µg) were run on 12% SDS-PAGE gel and then transferred electrophoretically to a polyvinylidene fluoride membrane (Millipore, Shanghai, China). The blots were blocked for 2 h at 25℃ with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST), followed by incubation with primary antibodies, including anti-JNK, anti-p-JNK, anti-caspase-3, anti-Bcl-2, anti-LC3, anti-p62, and anti-β-actin antibodies (Proteintech Group, Inc., Wuhan, China) at 4℃ overnight. Subsequently, membranes were washed with TBST five times for 5 mins each. Then, respective horseradish peroxidase-linked secondary antibodies (Beyotime, Shanghai, China) were added and incubated for 2 h at 37℃, followed by washing with TBST. Lastly, ECL solution was applied to the membrane evenly prior to detection of chemiluminescence (Thermo Scientific, Shanghai, China).