Bca protein kit

The cell lysates extracted from the skin tissues of mice and the cultured cells were used for immunoblotting analysis. Total protein concentration was measured using the BCA protein kit (Vazyme, China) with bovine serum albumin (BSA, Sigma-Aldrich) as the standard protein. Equal amounts of protein from each sample were subjected to 10% SDS PAGE gels electrophoresis, and subsequently transferred to PVDF membranes (Millipore). The membranes were blocked with 5% milk at room temperature for 1 h. Then the membranes were incubated with rabbit anti-HSP47 monoclonal antibody (1:1000) (Abcam, Hong Kong, Ltd.), rabbit anti-alpha-SMA monoclonal antibody (1:50000) (Millipore, clone E184), goat anti-Collagen type I polyclonal antibody  (1:500) (Millipore), rabbit anti-mouse anti-Collagen type I polyclonal antibody (1:500; Millipore) or internal control GAPDH antibody (Cell Signaling Technology) at 4°C overnight. After three washes with TBST for 30 min, the PVDF membranes were incubated with the horseradish peroxidase-conjugated secondary antibody of goat anti-rabbit, rabbit anti-goat or goat anti-mouse lgG for 1 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence system (Thermo) and the intensity of bands was quantified using ImageQuantTL software (General Electric Company).

Cell lysates extracted from murine lung tissue and cultured cells were used for subsequent immunoblot analyses. Total protein concentration was measured using a BCA protein kit (Vazyme, China). Equal amounts of protein from each sample were subjected to 10% SDS PAGE gels electrophoresis after which protein was transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked in 5% milk in TBST at room temperature for 1 h after which they were incubated with one of the following primary antibodies: mouse monoclonal [2A12] to 3-Nitrotyrosine (1:1000) (Abcam, Hong Kong, Ltd.), rabbit anti-mouse anti-Collagen type I polyclonal (1:500) (Millipore), or internal control GAPDH (1:5000–10000) (Vazyme, China) at 4 °C overnight. Membranes were washed three times with TBST for a total of 30 minutes and then incubated with the horse-radish peroxidase-conjugated secondary antibody of goat anti-rabbit, rabbit anti-goat, or goat anti-mouse lgG for 1 h at room temperature. The protein bands were visualized with ECL solution.