Ab134906

RIPA containing PMSF and phosphotransferase inhibitors (Beyotime Technology, Jiangsu, China) was used for protein extraction. Protein concentration was measured using a BCA protein assay kit (Beyotime Institute of Biotechnology). Equivalent protein concentrations were separated using SDS‐PAGE (12. 5%) and transferred onto PVDF membranes, which were subsequently blocked with 5% nonfat milk and incubated with relevant primary antibodies at 4°C overnight. Horseradish peroxidase‐conjugated secondary antibodies were used to detect primary antibodies. The ECL detection system (Millipore, Billerica, MA, USA) was used to image protein bands. Primary antibodies included anti‐β‐actin (1:1,000, ab8226, Abcam, Cambridge, MA, USA), anti‐Shh (1:1,000，ab53281，Abcam), anti‐Gli‐1 (1:1,000, ab134906, Abcam), anti‐Ptch1 (1:500, ab53715, Abcam), and anti‐Smo (1:1,000, ab72130, Abcam).

After cells were disrupted, the total protein from cells was extracted. Protein quantification was performed using a BCA kit, and an SDS/PAGE was conducted. The protein from SDS/PAGE gel was transferred onto a poly(vinylidene difluoride) (PVDF) membrane. After this, the PVDF membrane was blocked with 5% nonfat dry milk for 2 h at room temperature. The primary antibodies we used in our research were incubated at a temperature of 4 °C overnight, whereas PVDF membranes were rinsed three times by PBST. The antibodies of Gli1 (ab134906), SMO (ab113438), PTCH1 (ab53715) and HHIP (ab39208) were provided by Abcam. After PVDF membranes were washed once more, they were incubated with the corresponding secondary antibody, whereas the membranes were washed three times with PBST. Finally, an enhanced ECL chemiluminescent kit was used to treat the membrane for color reaction.

Total protein from freshly-frozen tissues was extracted by RIPA (Beyotime, China) containing a protease inhibitor mixture (KangChen, China). Protein concentrations were quantified using a BCA Protein Assay Kit (Pierce Biotechnology). Protein samples were fractionated on 10% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore). After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies at 4°C overnight. The membranes were labelled with HRP-conjugated secondary antibodies for 1 h at room temperature and the immunoreactive signals were detected using Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA).
The primary antibodies were NOX4 (1:1000, ab133303, Abcam), GLI1 (1:1000, ab134906, Abcam), GAPDH (1:1000, WeiAo, China China). Other antibodies were all purchased from Cell Signaling Technology Inc (USA). Secondary antibodies were HRP-conjugated goat anti-rabbit antibody (1:5000, WeiAo, China).

Western blot was carried out according to the protocols previously reported [32 (link)]. Total protein from cells were extracted using RIPA lysis buffer (Solarbio, Beijing, China). The protein concentration was determined with the application of a BCA assay kit (Beyotime, Jiangsu, China). Afterward, SDS-PAGE was prepared to separate proteins, and then the latter were transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, MA, USA). PVDF membranes were incubated with primary antibodies, including anti-SHH (1:1000, ab53281, Abcam, Cambridge, UK), anti-Gli1 (1:1000, ab134906, Abcam, Cambridge, UK), anti-CUL3 (1:1000, ab75851, Abcam, Cambridge, UK), anti-Nrf2 (1:1000, ab62352, Abcam, Cambridge, UK), anti-NQO1 (1:1000, ab80588, Abcam, Cambridge, UK) and Anti-GAPDH (1:1000, ab8245, Abcam, Cambridge, UK), and were removed the second day. In addition, secondary antibodies (Sigma-Aldrich) were prepared for incubation with PVDF membranes. The protein bands were visualized and analyzed using a chemiluminescence system (Bio-Rad, CA, USA).

RIPA lysis buffer was first employed to lyse the indicated cells, and then the collected total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (12%) and shifted to polyvinylidene difluoride membranes. After being blocked by 5% nonfat milk, the membranes were probed all night at 4°C with primary antibodies, including the loading control GAPDH (ab8245, 1/1000; Abcam, Cambridge, MA, USA), Nanog (ab109250, 1/1000; Abcam), OCT4 (ab181557, 1/1000; Abcam), N-cadherin (ab76057, 1/1000; Abcam), E-cadherin (ab76055, 1/1000; Abcam), CD9 (ab92726, 1/2000; Abcam), HSP70 (ab2787, 1/1000; Abcam), CD81 (ab109201, 1/1000; Abcam), TSG101 (ab125011, 1/1000; Abcam), α-smooth muscle actin (α-SMA) (#19245 S, 1/1000; Cell Signaling Technology, Danvers, MA, USA), fibroblast activation protein (FAP) (#66562 S, 1/1000; Cell Signaling Technology), SMO (ab8969, 1/1000; Abcam), Gli1 (ab134906, 1/1000; Abcam) and collagen type I (ab34710, 1/1000; Abcam). After processing with tris-buffered saline and polysorbate 20 thrice, the membranes were subjected to cultivation with corresponding horseradish peroxidase-labeled secondary antibodies at room temperature for 2 h. The protein signals were examined using the ECL luminous liquid (Pierce, Rockford, IL, USA). Western blot was conducted in triplicate three times.

Western blotting was performed according to a previously described protocol (35 (link)). Briefly, cells in a 6-well plate were lysed on ice with RIPA buffer containing protease inhibitors, and the lysates were centrifuged at 12,000 rpm at 4°C for 10 min. The protein concentration was measured using the BCA Protein Assay Kit (23227; Thermo Fisher Scientific, Waltham, MA, USA). Then proteins were resolved on 10% SDS-PAGE gels and electrotransferred to the Pure Nitrocellulose Blotting Membrane (Pall Corporation, Port Washington, NY, USA). The membrane was blocked in 5% milk, and incubated overnight at 4°C with the following primary antibodies: RAB31 (1:400, 16182-1-AP; Proteintech, Rosemont, IL, USA), GLI1 (1:1,000, ab134906; Abcam, Cambridge, MA, USA), cyclin D1 (1:5,000, ab134175; Abcam), c-Myc (1:1,000, ab39688; Abcam), B-cell lymphoma 2 (Bcl-2) (1:1,000, ab32124, Abcam), and Bcl-2-associated X protein (BAX) (1:1,000, ab32503, Abcam). After washing, the membrane was incubated at room temperature for 1 h with the same secondary antibodies as those used in our previous study (1:3,000; WeiAo Pharmaceutical, Sichuan, China) (35 (link)). The protein signals were detected using the ChemiDoc^TM Imaging System (Bio-Rad, Hercules, CA, USA).