Ab0037

NSCLC tissue samples were obtained from 8 NSCLC patients and matched with adjacent normal tissue samples for Western blot analysis. NSCLC cells were trypsinized and centrifuged. The pellets and tissue samples were resuspended in cell lysis buffer (P0013B, Beyotime Institute of Biotechnology) that contained 1% protease inhibitor (ST506; Beyotime Institute of Biotechnology) separately. Approximately 30 μg of proteins per lane were separated in 15% sodium dodecyl sulphate gels by polyacrylamide gel electrophoresis. The gels were transferred to polyvinylidene fluoride membranes (IPVH00010; Millipore Corporation), and the membranes were blocked with TBST containing 5% nonfat milk powder. Subsequently, the membranes were incubated with a primary antibody against CHP2 (1:500 dilution, GTX87522; Genetex) at 4°C overnight. Following washing with TBST, the membranes were incubated with secondary horseradish peroxidase–conjugated goat anti-rabbit IgG (1:2000, ab205718; Abcam) for 2 hours at room temperature. Immunoreactive proteins were evaluated using an enhanced chemiluminescence system (Bio-Rad, Hercules, CA). Glyceraldehyde 3-phosphate dehydrogenase (1:2000, AB0037; Abways) was used as the internal control sample. Signal intensities were subsequently quantified using the Image J quantification software.

Lysates from ovarian tissue and mGCs were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were incubated with MTR antibody (1:2000, ab66039, Abcam, UK), BHMT antibody (1:200, ab243698, Abcam, UK), LC3B antibody (1:1000, ab48394, Abcam, UK), P62 antibody (1:1000, F032602, Abways Technology), Beclin-1 antibody (D40C5, 1:500, 3495s, Cell Signaling Technology, Inc.), mTOR (s2442) antibody (1:1000, BS3611, bioWORLD), and p-mTOR antibody (1:1000, BS4706, bioWORLD). In addition, samples were incubated with peroxidase-conjugated secondary antibody, and an enhanced chemiluminescence detection kit (Amersham Pharmacia Biosciences, Inc.) was used for visualization. Protein levels were quantified and normalized using glyceraldehyde 3-phosphate dehydrogenase (1:10,000, AB0037, Abways Technology) as internal control.

To verify the expression of KGF, Ang-1(angiopoietin-1), β-catenin, NGFR, ERK, and other proteins, cellular proteins were extracted from MSCs after exposure to different concentrations of ISX-9. Protein concentrations were determined using the BCA assay (CST, #7780, USA). Proteins were separated on SDS-PAGE gels prior to transfer to PVDF membranes (Life Technologies, USA). PVDF membranes were closed in a fast closure solution for 30 minutes before being incubated overnight with various primary antibodies, including KGF (Abcam, ab131162), angiopoietin 1 (Ang-1, Abcam. ab102015, UK), p-β-catenin (Affinity, DF2989), β-catenin (Affinity, BF8016), TAU (Affinity, AF6141), p-TAU (Abways, CY5657), NGFR (Abcam, ab52987), cleaved caspase-3 (Affinity, AF7022), cleaved caspase-9 (Affinity, AF5240), Bax (Wanleibio, WL03446), GAPDH (Abways,AB0037) were used as controls for total proteins. The membranes were then incubated with HRP secondary antibody (Jackson ImmunoResearch Laboratories, 111-035-003, 115-035-003, China). Blots were detected by the ECL technique (115-035-003, China).

Five mouse bladders of each group were homogenized with RIPA buffer (Beyotime Biotechnology, P0013B). Following 30 min reaction on ice, the tissue debris was pelleted by centrifugation (14,000g, 15 min, 4 °C). The supernatant was collected, and its concentration was determined by using bicinchoninic acid (BCA, Beyotime Biotechnology, P0010). Protein samples were heated for 10 min to 99 °C with loading buffer. Protein samples were separated to SDS-PAGE and were transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk for 1.5 hours at room temperature and incubated overnight at 4 °C with primary antibodies (TRPV1, 1:1,000, ABCAm #ab203103). GAPDH (1:2,000, Abways #AB0037) was used as the referenced protein. After three washing times with Tris-buffered saline with tween-20 (TBST) for 10 min each, membranes were incubated with HRP-conjugated secondary antibodies (1:5,000, Cell Signaling Technology, #7076) for 1 hour at room temperature and washed three times with TBST. Signaling was detected with Amersham ImageQuant 800.