Ecl western blot detection system

Proteins (20 µg) were separated by SDS-polyacrylamide gel electrophoresis and blotted to polyvinylidene fluoride membranes (Bio-Rad, Munich, Germany). Membranes were incubated with the antibodies in 1.5% BSA/PBS/0.1% Tween and detection of the immune complexes was performed with the ECL Western blot detection system (Amersham Pharmacia, Deisenhofen, Germany). The antibodies used were obtained from the following sources: human chemerin, R&D Systems (Wiesbaden-Nordenstadt, Germany); GAPDH, New England Biolabs GmbH (Frankfurt am Main, Germany); galectin-3, BD Biosciences (Heidelberg, Germany); α-SMA and CMKLR1, Abcam (Cambridge, UK); GPR1, Bioss (Woburn, MA, USA) and Abnova (Taipei City, Taiwan). ImageJ software was used for quantification [43 (link)].

To prepare whole-cell lysates, the cells were washed twice with cold PBS and harvested. The cells were lysed with 0.1% NP-40-added RIPA buffer (Thermo Fisher) [51 (link)]. Protein assays were performed using the Protein Assay Rapid Kit (Wako). Protein lysates (25 μg) were separated on 12.5% sodium dodecyl sulfate-polyacrylamide gels, followed by electrotransfer onto a nitrocellulose filter. The membranes were incubated with primary antibodies and then with peroxidase-conjugated IgG antibodies (Agilent Technologies, Santa Clara, CA, USA). Immune complexes were detected using an ECL Western blot detection system (Amersham, Aylesbury, UK). The following primary antibodies were used, at a working dilution of 1:1000, for immunoblot analyses: antibodies against c-Myc (#18583), PGC-1α (#4259), SLC7A11 (#98051), Parkin (#2132), PINK1 (#6946), LC3A/B (#4108), ATG5 (#12994) (Cell Signaling Technology Japan, Tokyo, Japan), ACSL4 (bs-13129R), GPX4 (bs-3884R) (Bios Antibodies, Woburn, MA, USA), and β-actin (ab178787), GAPDH (ab8245, Abcam).

Whole-cell lysates were prepared as previously described [66 (link)]. Protein lysates (25 μg) were separated on 12.5% sodium dodecyl sulfate-polyacrylamide gels, followed by being electrotransferred onto a nitrocellulose filter. The membranes were incubated with primary antibodies, and then with peroxidase-conjugated IgG antibodies (Agilent Technologies, Santa Clara, CA, USA). Immune complexes were detected using an ECL Western blot detection system (Amersham, Aylesbury, UK). The following primary antibodies were used, at a working dilution of 1:1000, for immunoblot analyses: antibodies against CD44 (c-9960), CD47 (sc-7059), nucleostemin (NS) (sc-166460), MDR (sc-13131), RELA (sc-372), β-actin (sc-47778) (Santa Cruz Biotechnology Inc., Dallas, TX, USA), E-cadherin (Abcam, Cambridge, MA, USA; ab1416), snail (#3879), and phospho-Akt (Ser473) (#4060) (Cell Signaling Technology Japan, Tokyo, Japan).

To examine whether everolimus or sunitinib affects the mTOR pathway, Caki‐1 cells were cultured in RPMI 1640 containing 10% FBS for 24 h and incubated for 2 days in medium with or without everolimus (20 nmol/L) or sunitinib (20 μmol/L).
To examine whether these drugs affect the PDGF pathway, Caki‐1 cells were cultured in serum‐free culture medium for 1 h and then stimulated with 10 ng/mL PDGF‐BB for 15 min. Cells were harvested by scraping in Tris‐glycine sodium dodecyl sulfate sample buffer (Invitrogen). The proteins of harvested cells were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose filters. The immune complexes generated following hybridization with anti‐PDGF‐Rβ (1:50) or anti‐pPDGF‐Rβ (1:200) primary antibodies were visualized by enhanced chemiluminescence with an ECL Western Blot Detection System (Amersham Biosciences, Piscataway, NJ). β‐Actin (Sigma) was also stained as a loading control.

In order to detect various proteins from cells and spheroids, a proper amount of samples were homogenized and digested in 1× RIPA buffer (Cell Signaling, Denver, MA, USA) which includes 1 mM PMSF (Fluka, Switzerland), 2 g/mL Aprotinin (Sigama, Steinheim, Germany), 1 mM DTT(Invitrogen, Carlsbad, CA, USA) and phosphatase inhibitor cocktail solution (GenDEPOT, Barker, TX, USA) on ice for 1 hr. After the digestion, samples were centrifuged for 25 min at 14,000 rpm in cold microcentrifuge. Then supernatants were collected for use. Western blot experiments were performed following the standard protocol. The following primary antibodies were purchased: anti-YAP/TAZ (8418, Cell Signaling) and anti-EpCAM (ab71916, Abcam). anti-EpCAM was used and for internal control, an anti-GAPDH antibody (2118, Cell Signaling) was used. Anti-rabbit IgG-HRP (A0545, Sigma) was used as the secondary antibody. Specific bands were detected using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

A standard Western Blott protocol was used as previously described^{18 (link)}. hPDL-derived cells (in basal media) were harvested using trypsin/EDTA (Gibco), washed twice with PBS, resuspended in RIPA lysis buffer (Millipore) in the presence of protease inhibitors (Pierce^TM. protease inhibitor Mini Tables, Pierce Biotechnology Inc) and PMSF 1 M (Abcam) for 30 min at 4 °C. Protein concentration was determined using the bradford protein assay (Sigma-Aldrich). Proteins were separated in 8% SDS-polyacryamide gel (PAGE-SDS) and transferred to a nitrocellulose membrane (Whatman). PageRuler™ Prestained Protein Ladder (Thermo Scientific) has been used as size standards in protein electrophoresis (SDS-PAGE) and Western-Blotting. After transfer, nitrocellulose membranes were stained with Ponceau S solution (Sigma-Aldrich) to visualize protein bands. Blots were then incubated over-night at 4 °C with rabbit antibody against β-III-tubulin (TUJ1; 1:1000, Covance). Secondary antibody was used at 1:7000 for peroxidase anti-mouse Ab (PI-2000, Vector Laboratories). Immunoreactivity was detected using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham Biosciences Europe) and Luminata^TM Forte (Millipore corporation) using ImageQuant LAS 500 Gel Documentation System (GE Healthcare). The molecular weight of β-III-tubulin is approximately 55 kDa.