Anti rabbit irdye 800

Whole cell lysates were prepared by lysis buffer (50mM Tris-HCl, 150mM NaCl, 1% Triton-X 100, 1mM MgCl₂, MnCl₂ and CaCl₂, 1mM PMSF and 10mM sodium fluoride) [49 (link)]. The primary antibodies used included the following: DPT (HuaAn Biotechnology, Hangzhou, China), focal adhesion kinase (FAK) (Abcam, Cambridge, UK), p-FAK Tyr397 (Abcam, Cambridge, UK), Src (Cell Signaling Technology, Boston, MA), p-Src Tyr527 (Cell Signaling Technology, Boston, MA), ITGA3 (Abcam, Cambridge, UK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech Group, Chicago IL). After incubating with the IRDye 680 anti-mouse (LI-COR, Lincoln, NE) and IRDye 800 anti-rabbit (LI-COR, Lincoln, NE) secondary antibodies for 1 hour at room temperature, the bands were detected by an Odyssey infrared imaging system (LI-COR, Lincoln, NE). Quantification was analyzed by using Image J software.

Cells lysates were prepared by RIPA buffer containing protease inhibitors. Denatured lysates were separated by 10% SDS-PAGE gel electrophoresis and transferred onto PVDF membranes (Millipore Corp, Billerica, MA, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies overnight at 4°C. The following antibodies were used: anti-TUFT1 (1: 500 dilution, Santa Cruz), anti-phospho-CREB1 (Ser133, 1 : 1000, ImmunoWay), anti-ZYX (1 : 1000, Proteintech), and anti-β-actin (1 : 1000 dilution, Santa Cruz). After washing with TBS/Tween-20, the membranes were incubated with IRDye 680 anti-mouse or IRDye 800 anti-rabbit (LI-COR) secondary antibodies for 1 hr at room temperature. The bands were detected by Odyssey imaging system (LI-COR).

Docetaxel and sulforhodamine B were purchased from Sigma-Aldrich (St. Louis, MO). Cabazitaxel (Sanofi, Bridgewater, NJ) was a generous gift from Dr. Robert Nagourny (Rational Therapeutics, Long Beach, CA). Ellagic acid was obtained from Indofine Chemical (Hillsborough, NJ). Urolithin A was obtained from Santa Cruz (Dallas, TX). Protocatechuic acid was purchased from LKT Labs (St. Paul, MN). Purified tubulin protein was purchased from Cytoskeleton (Denver, CO). Phenol-red free matrigel was obtained from Corning (Bedford, MA). DMSO, ethanol, and trichloroacetic acid were obtained from Thermo Fisher Scientific (Waltham, MA). Antibodies against β-actin (catalog #3700), β-tubulin (#2128), anti-mouse Alexa Fluor 488 (#4408), and anti-rabbit Alexa Fluor 555 (#4413) were obtained from Cell Signaling Technologies (Danvers, MA). GAPDH (#365062) antibody was obtained from Santa Cruz Biotechnology (Dallas, TX). IRDye 800 anti-rabbit (#926-32211) and IRDye 680 anti-mouse (#926-68070) antibodies were obtained from Li-Cor Biotechnology (Lincoln, NE).

Total cellular proteins were extracted using a total protein extraction kit (Beyotime, China). Cell lysates were separated by 10% SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were blocked with 5% nonfat milk and incubated with the primary antibodies and then incubated with species-specific secondary antibodies. Image J software was used to analyze the results. The following antibodies were used at the indicated concentrations PEA15 (1:1000, Proteintech, USA), Bcl2 (1:1000, CST, USA), Bax (1:1000, CST, USA), Cleaved caspase-3 (1:1000, CST, USA), ERK(1:1000, Abcam, USA), p-ERK(1:1000, Abcam, USA), IRDye680 anti-mouse (1:20000, LI-COR) and IRDye800 anti-rabbit (1:10000, LI-COR). GAPDH (1:1000, CST, USA) was used as a control to confirm equal loading of protein samples.

[³H]-TPP (specific activity: >1.4 Ci/mmol; radiochemical purity: >98.2%) and [³H]-thiamin (specific activity: >12.8 Ci/mmol; radiochemical purity: >93.3%) were purchased from Moravek, Inc. Human-specific anti-TPPT (SLC44A4) affinity-purified rabbit polyclonal antibody was generated for us by Thermo Fisher Scientific; anti-THTR-1 (catalog no.: ab229680) rabbit polyclonal antibody was from Abcam; anti-THTR-2 (catalog no.: 13407-1-AP) rabbit polyclonal antibody was from Proteintech; anti-HIF-1α (catalog no.: 14179S) rabbit monoclonal antibody was from Cell Signaling Technology; anti-HIF-2α (catalog no.: NB100-122) rabbit polyclonal antibody was from Novus Biologicals; and anti-GAPDH mouse monoclonal antibody (catalog no.: sc-47724) was from Santa Cruz. The secondary antibodies, anti-rabbit IRDye-800 (catalog no.: 926-32211) and antimouse IRDye-680 (catalog no.: 926-68020), were purchased from LI-COR Bioscience. All other chemicals and reagents used in this studies were of analytical/molecular biology grade and purchased from established sources.

NCM460 cells were from INCELL (San Antonio, TX), and [³H]-TPP (specific activity 1.3 Ci/mmol; radiochemical purity 97%) was from Moravek Biochemicals (Brea, CA); qPCR primers were from Sigma Genosys (Woodlands, TX); Other chemicals/reagents were from commercial vendors and were of analytical/molecular biology grade. Human and mouse specific anti-SLC44A4 polyclonal antibodies were generated for us by Alpha Diagnostics International (San Antonio, TX) and by Thermofisher (Rockford, IL), respectively. Human anti-ELF3 (AV31639) antibody was from Sigma-Aldrich (Saint Louis, MO); anti-CREB-1 (#9197), anti-ERK1/2 (#9102S), anti-Phospho ERK1/2 (#9101S) and anti-Phospho NF-κB (#3033S) were from Cell Signaling Technology (Danvers, MA); anti-NF-κB (ab16502) antibody from Abcam (Cambridge, MA); anti-β-actin (sc-47778) and anti-Lamin B (sc-6216) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibodies, anti-rabbit IRDye-800 and anti-mouse IRDye-680, were purchased from LI-COR Bioscience (Lincoln, NE).