α sma 55135 1 ap

Target tissue samples or cells were homogenized in ice-cold RIPA lysis buffer containing 1% protease inhibitor for 30 min. The lysates were centrifuged, and the supernatants were then recovered. The protein concentrations were measured with a bicinchoninic acid protein assay kit. The protein samples were then applied for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to a polyvinylidene fluoride membrane. The membranes were firstly blocked with 5% non-fat milk and then hybridized with the following antibodies: α-SMA (55135-1-AP; Proteintech, Wuhan, China), collagen I (66761-1-Ig, Proteintech), E-cadherin (20874-1-AP, Proteintech), vimentin (10366-1-AP, Proteintech), OGN (A07061, Boster, Pleasanton, CA, USA), p-mTOR (ab109268, Abcam), mTOR (ab2732, Abcam), Akt (Y409094, ABM, New York, NY, USA), p-Akt (Y011054, ABM) at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies and ECL were used for detection and the protein expression was normalized to that of β-actin.

IHC staining of colorectal tissues was performed on 5-µm-thick formalin-fixed and paraffin-embedded (FFPE) tissue sections according to the standard procedures as described previously [14 (link)]. The sections were stained using antibodies against CXCR7 (ab72100) (Abcam, USA), α-SMA (55135-1-AP), Vimentin (10366-1-AP), and Ki67 (27309-1-AP) (Proteintech, USA) according to the manufacturers’ instructions. The IHC results were taken with a digital slide scanning system (Pannoramic Scan, 3DHISTECH Ltd) and semiquantified of mean density (IOD/area) by image-pro plus 6 software (IPP, USA).

RIPA Buffer (Tris–HCl 25 mM pH 7.4, NaCl 150 mM, 1% Triton X-100, Sodium Deoxycholate 1%, SDS 0.1%, EDTA 2 mM) containing a protease/phosphatase inhibitor mixture (Roche) was utlized to lyse cells. After electrophoresis with SDS-PAGE, the separated proteins from the gel (50 μg) were transferred on nitrocellulose (NC) membranes, followed by incubation with primary antibodies. Following incubation with appropriate HRP-labeled secondary antibodies, ECL HRP substrate (Beyotime) was employed to detect signals. The primary antibodies used were as follow: α-SMA (55135-1-AP, Proteintech), Collagen I (14695-1-AP, Proteintech), Timp-1 (CSB-PA023560YA01HU, CUSABIO, Wuhan, China), Vimentin (10366-1-AP, Proteintech), Erk (16443-1-AP, Proteintech), p-Erk (sc-81492, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p38 (14064-1-AP, Proteintech), p-p38 (AF4001, Affinity, Changzhou, China), JNK (AF6319, Affinity), p-JNK (AF3318, Affinity), NF-κB (AF5006, Affinity), p-NF-κB (AF2006, Affinity).