Sc 420

Artery samples were fixed in 4% paraformaldehyde solution for paraffin embedding. Frozen tissues were embedded in the O.C.T. compound (Tissue-Tek). Sections were blocked in 5% goat serum for 1 h at room temperature, then incubated with primary antibody at 4 °C overnight. The primary antibodies used in this study was as follows. CD31 (1:100, #ab28364, Abcam; 1:100, #ab9498, Abcam), caveolin-1 (1:50, #3267, Cell Signaling Technology), Sp1 (1:50, #sc−420, Santa Cruz Biotechnology Inc.), Sp3 (1:50, #sc-28305, Santa Cruz Biotechnology Inc.), p-AMPKα (Thr172, 1:100, #50071, Cell Signaling Technology). After washing with phosphate buffered saline, samples were incubated with Alexa Fluor-labeled secondary antibody at room temperature for 1 h including Alexa Fluor 488 (1:200, #ab150077, Abcam; 1:200, #ab150113, Abcam), Alexa Fluor 594 (1:200, #ab150080, Abcam; 1:200, #ab150116, Abcam). Finally, nuclei were stained with 4,6’-diamidino-2-phenylindole (DAPI, #ab104139, Abcam) for 5 min at room temperature, and immunofluorescence was analyzed under a florescent microscope.

The murine lung tissue samples were made into homogenate and centrifuged at 20,000 at 4°C for 30 min to collect total protein, and the protein concentration was determined using the bicinchoninic acid method (23225, Pierce Biotechnology, Waltham, MA, USA). Then, the protein sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with the primary antibodies against SP1 (sc-420, 1:1,500, Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA), PTEN (MA5-12,278, 1:2,000, Thermo Fisher Scientific), AKT (sc-5298, 1:1,500, Santa Cruz), p-AKT (NB100-56749, 1:1,000, Novus Biologicals, Littleton, CO, USA) and GAPDH (ab8245, 1:2,000, Abcam) at 4°C for 16 h, and then with the secondary antibody (ab205719, 1:5,000, Abcam) at 37°C for 3 h. The protein bands were analyzed using an enhanced chemiluminescence (ECL) kit (PE0010, Solarbio) [31 (link)].

Cells were collected and proteins were obtained using RIPA. BCA kit (Thermo Scientific, MA, USA) was used to ensure the concentrations. Total protein were separated with SDS-PAGE and transferred to NC membranes (Merck millipore, USA) later. Then membranes were blocked in 5% milk without fat in TBST and incubated with primary antibodies at 4 ℃ for 16 h. After being washed with TBST for three times, the blots were then incubated with secondary antibody for 2 h at room temperature. The blots were developed using ECL reagent. The following antibodies were used: Sp1 (sc420; Santa cruz; 1:1000), TIMP1 (ab211926; Abcam; 1:1000), ACTB (AC004; Abclonal; 1:3000), anti-mouse HRP (31430; Thermo; 1:3000), anti-rabbit HRP (31460; Thermo; 1:3000).