Bca method

RIPA buffer (CST, USA) was used to extract the total protein, followed by the quantification based on the BCA method (Abbkine, USA). The proteins at same concentration were subjected in SDS-PAGE and transferred (PVDF, Millipore, USA), followed by the incubation with 5% milk and with the primary antibodies at 4° C overnight. The corresponding second HRP-conjugated anti-mouse or anti-rabbit antibodies (Boster, Wuhan, China) were used for incubating the membranes 1 hour at room temperature, followed by the visualization by using chemiluminescence detection kit (Beyotime, Shanghai, China). The primary antibodies applied in this study comprise of patched-1 (Affinity, USA), Gli-1 (Affinity, USA), PI3K (Affinity, USA), Akt (Affinity, USA), eNOS (Affinity, USA), p-Akt (Affinity, USA), and β-actin (Abcam, USA). All antibodies were diluted in PBST solution at 1:2000 (v/v).

RIPA buffer (CST, USA) was used to extract the total protein, followed by the quantification based on the BCA method (Abbkine, USA). The proteins at same concentration were subjected in SDS-PAGE and transferred (PVDF, Millipore, USA), followed by the incubation with 5% milk and with the primary antibodies at 4°C overnight. The corresponding second antibodies (Boster, China) were used for incubating the membranes 1 hour at room temperature, followed by the visualization by using chemiluminescence detection kit (Beyyotime, China). The primary antibodies applied in this study comprising Bax (1:2000, ab32502, Abcam, USA), Bcl-2 (1:2000, ab32124, Abcam, USA), caspase-3 (1:2000, ab32351, Abcam, USA), and β-actin (1:2000, ab 8226, Abcam, USA). The grayscale of the images was quantified and calculated using a gel image system (Bio-Rad, USA) and the relative level of each band was normalized to the level of β-actin.

RIPA buffer (CST, USA) was used to extract the total protein, followed by the quantification based on the BCA method (Abbkine, USA). The proteins at same concentration were subjected in SDS-PAGE and transferred (PVDF, Millipore, USA), followed by the incubation with 5% milk and with the primary antibodies at 4° C overnight. The corresponding second antibodies (BOSTER, China) were used for incubating the membranes 1 hour at room temperature, followed by the visualization by using chemiluminescence detection kit (Beyyotime, China). The primary antibodies applied in this study comprising SOX4 (Abcam, USA), collagen II (Abcam, USA), aggrecan (Abcam, USA), MMP13 (Abcam, USA), ADAMTS4 (Abcam, USA), Bax (Abcam, USA), caspase3 (Abcam, USA), cleaved-caspase3 (Abcam, USA), caspase9 (Abcam, USA), cleaved-caspase9 (Abcam, USA), β-actin (Abcam, USA).