Enhanced bca protein kit

HepG2 cells were cultured in line with the above. Nuclear and total cellular proteins were extracted using commercial kits (Beyotime, China). Protein concentrations were also determined using an enhanced BCA protein kit (Beyotime, China). Cellular protein samples were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then electrotransferred to polyvinylidene fluoride (PVDF) membranes. Blocking was performed for 40 min using Protein Free Rapid Blocking Buffer (Yamei, China), and primary antibody blocking was performed overnight at 4 °C. The membrane was then washed three times with T-TBST for 10 min each and then bound to the secondary antibody for 1 h at room temperature. After three more T-TBST washes, the kit was detected using an ECL kit with an automated chemiluminescence image analysis system (BIO-RAD, America). The western blots were analyzed using ImageJ software (NIH, Bethesda, MD, USA). And the blots were cut prior to hybridisation with antibodies during blotting.

Exosome protein was obtained using the RIPA Lysis Buffer (Beyotime, China). The protein concentrations were determined with the Enhanced BCA Protein Kit (Beyotime) [51 (link)]. After SDS polyacrylamide gel electrophoresis and electroblotting, the membranes were incubated with the following primary antibodies: anti-GAPDH mouse monoclonal antibody (Abcam, UK), anti-calnexin monoclonal antibody (Cat#: 66903-1-Ig, Proteintech), anti-Alix monoclonal antibody (Cat#: 12422-1-AP, Proteintech), and anti-TSG101, CD9 rabbit polyclonal antibody (Cat#: 67381-1-Ig, Cat#: 20597-1-AP, Proteintech). HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) and HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (Cat#: SA00001-2, Cat#: SA00001-1, Proteintech) were used. Blots were developed using the Tanon ABL X5 Series Intravital Imaging Systems (Tianneng Technology, China) and quantified with ImageJ software.

The skin and cell samples were collected for detecting the protein expression level. Protein lysates were obtained using the RIPA lysis buffer (PPLYGEN, Beijing, China). The protein concentration was measured using the Enhanced BCA Protein Kit (Beyotime, Shanghai, China). The protein samples were diluted to 0.5 μg/μL for detecting the protein level. The protein (1.5 ng) was analyzed using the automated Western blotting system (Protein Simple Wes) [29 ], according to the manufacturer’s instructions. The following antibodies were used: 1:2000 anti-GAPDH mouse monoclonal antibody (Abcam), anti-WNT2 rabbit polyclonal antibody (Bioss, Beijing, China), and 1:100 anti-CCND1 mouse monoclonal antibody, anti-LEF1 rabbit polyclonal antibody, anti-KRTAP11-1 mouse polyclonal antibody, and anti-STAT1 mouse monoclonal antibody (Proteintech, Wuhan, China).

The rabbit skins were collected for estimating the protein expression level. RIPA lysis buffer (PPLYGEN, Beijing, China) was used for collecting protein lysates, and the protein concentration was measured using the Enhanced BCA Protein Kit (Beyotime, Shanghai, China). According to the manufacturer’s instructions, 0.5 μg/μL of the protein sample was used for detecting the protein expression level by using the automated Western blotting system (Protein Simple Wes) [48 ]. The figures of Wes analysis were showed in Supplementary file S1. The following antibodies were used: 1:100 anti-CCND1 mouse monoclonal antibody, anti-LEF1 rabbit polyclonal antibody (Proteintech, Wuhan, China), and 1:100 anti-GAPDH mouse monoclonal antibody (Abcam, Cambridge, MA, USA).