Rabbit anti human nf κb1 p50 ab

IM-0223 cells (2 × 10⁴ cells) were incubated in 8-well chamber slides (Thermo Fisher Scientific), and stained for immunofluorescence as previously described (28 (link)), using the following Abs: mouse anti-human KPC1 Ab (1:50 dilution, #ab57549; Abcam), rabbit anti-human NF-κB1 p50 Ab (1:50 dilution, #sc-114; Santa Cruz Biotechnology), Alexa Fluor 488-conjugated goat anti-mouse IgG (2.5 μg/ml, #115-545-003, Jackson ImmunoResearch, West Grove, PA) or Cy3-conjugated goat anti-rabbit IgG (2.5 μg/ml, #111-165-003, Jackson ImmunoResearch). The slides were stained with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) in mounting medium. Images were obtained using a Nikon Eclipse Ti microscope and NIS elements software (Nikon).

Protein extraction and WB were performed as previously described (25 (link)). The following antibodies (Abs) were used: mouse anti-human KPC1 Ab (1:300, #ab57549; Abcam, Cambridge, MA), rabbit anti-human NF-κB1 p50 Ab (1:200, #sc-114; Santa Cruz Biotechnology, Dallas, TX), mouse anti-Myc-tag Ab (1:1000, #05-724; Millipore, Billerica, MA), mouse anti-Flag Ab (1:1000, #TA50011-100; OriGene), mouse anti-human β-actin Ab (1:10000, #A5441; Sigma-Aldrich), or horseradish peroxidase-conjugated Abs (sheep anti-mouse Ab (1:4000, #NA931; GE Healthcare, Pittsburgh, PA) or donkey anti-rabbit Ab (1:4000, #NA934; GE Healthcare)). Immunoreactive bands were visualized with the SuperSignal West Femto Maximum Sensitivity Substrate (Life Technologies), and the densities of protein bands were quantified using ImageJ software (http://imagej.nih.gov/ij/).