Irdye 800 conjugated affinity purified anti mouse igg

The primary antibodies were MTDH (ab45338), CD133, and LEF1 (Abcam, Cambridge, MA, USA), CD44, Oct4, CDH1/E-cadherin, and vimentin (Origene, Rockville, MD, USA), phosphorylated β-catenin and CTNNB1/β-catenin (Cell Signaling Technology; Danvers, MA 8480, USA). β-actin was obtained from ZSGB-bio (Beijing, China). IRDye 800-conjugated affinity purified anti-mouse IgG and IRDye 700DX-conjugated affinity purified anti-rabbit IgG secondary antibodies were purchased from Rockland Immunochemicals (Limerick, PA, USA) and CF666 anti-rabbit IgG secondary antibody was purchased from Biotium (Hayward, CA, USA).

Antibodies used in our study are as follows: mouse monoclonal anti‐DHFR (Abnova, Taipei, Taiwan), anti‐Cyclin B (Santa Cruz Biotechnology Inc., TX), anti‐MRE11 (GeneTex, Hsinchu, Taiwan), anti‐γH₂AX (Millipore, MA), anti‐CDK1 (Cell Signaling, Boston) and anti‐GAPDH (Kang Chen Bio‐tech, Shanghai, China); rabbit monoclonal anti‐BRCA1, anti‐RAD51 (Santa Cruz Biotechnology Inc., TX) and anti‐Histone H3 (Abcam, Cambridge, UK); CF488 goat anti‐mouse IgG and CF488 goat anti‐rabbit IgG (Biotium, CA); IRDye 800 conjugated affinity purified anti‐mouse IgG and IRDye 700 conjugated affinity purified anti‐rabbit IgG (Rockland, Philadelphia).

For immunoprecipitation, whole-cell extracts were incubated for 2 h at room temperature with rabbit affinity-purified anti-GFP antibodies (ab290; Abcam, Cambridge, MA) or antibodies against XMAP4 MBD bound to protein A agarose (Thermo Scientific). Agarose beads were pelleted and washed, and adsorbed proteins were eluted by the treatment of beads with SDS–PAGE sample buffer for 5 min at 100°C.
Immunoblotting was performed by the method described in Towbin et al. (1979 (link)). Blots were stained with mouse monoclonal GFP antibodies (ab1218; Abcam), p150^Glued subunit of the dynactin complex (610473; BD Biosciences, San Jose, CA), cytoplasmic dynein IC (74.1; Covance, Princeton, NJ), or kinesin-2 motor subunit (K2.4; Covance). Immunoreactive bands were detected with SuperSignal West Femto maximum-sensitivity substrate (Thermo Scientific, Waltham, MA). To measure the amounts of recombinant XMAP4, nonphosphorylatable and phosphomimetic mutant proteins cosedimented with MTs, blots of MT pellets were successively incubated with antibodies against XMAP4 MBD and IRDye800-conjugated affinity-purified anti-mouse IgG (Rockland Immunochemicals, Gilbertsville, PA), and the intensity of the infrared signal was quantified with the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE).