Octa probe

Whole cell extracts were prepared as described [2 (link)] and resolved by SDS-PAGE (between 8% and 12%) and transferred to polyvinylidene difluoride (PVDF) membranes. Samples were analyzed with the following antibodies: HDAC6 (H-300, Santa Cruz, Santa Cruz, CA, USA, and PA5–11240, ThermoFischer Scientific), HA (HA-7, Sigma–Aldrich), Acetylated α-tubulin (6–11B-1, Santa Cruz, Santa Cruz, CA, USA), α-tubulin (T5168, Sigma–Aldrich), β-Actin (I-19, Santa Cruz, Santa Cruz, CA, USA), RanBPM (5 M, Bioacademia, Japan), Rmnd5A (NBP1–92337, Novus Biologicals) and muskelin (C-12, Santa Cruz, Santa Cruz, CA, USA). The blots were developed using Clarity ECL Western Blotting Substrate (BioRad, Hercules, CA). Quantifications were done using Image Lab (BioRad, Hercules, CA) and ImageJ software. Co-immunoprecipitation experiments were performed in 0.25% NP-40 and 100 mM KCl lysis buffer and were carried out overnight at 4 °C with antibodies to HA (HA-7, Sigma–Aldrich), OctA-Probe (D-8 Santa Cruz, Santa Cruz, CA, USA), HDAC6 (D-11 Santa Cruz, Santa Cruz, CA, USA) and RanBPM (F1 Santa Cruz, Santa Cruz, CA, USA). Immunoprecipitates were isolated with PureProteome Protein G Magnetic Beads (EMD Millipore, Billerica, Massachusetts) or Dynabeads Protein G (Invitrogen, Life Technologies, Burlington ON, Canada).

Antibodies against E-cadherin, N-cadherin, and Matrigel basement matrix were purchased from BD Biosciences (San Jose, CA, USA). Antibodies against IGFBP-3, vimentin, myc, glutathione S-transferase (GST), His-probe, HA-probe, OctA-probe, IGF-1R, ubiquitin, and actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against E-cadherin, N-cadherin, and tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from GeneTex (Irvine, CA, USA). Fluorochrome (Alexa Fluor 488, Alexa Fluor 546, or Alexa Fluor 594)-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Human recombinant IGFBP-3 was purchased from R&D Systems (Minneapolis, MN, USA). Human recombinant IGFBP-3 protein used or evaluation of cellular uptake of extracellular IGFBP-3 was kindly provided by Insmed Inc. (Glen Allen, VA, USA) [22 (link)]. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from MP Biomedicals (Santa Ana, CA, USA). G418 was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Crystal violet, a mouse monoclonal anti-vimentin antibody, and additional chemicals unless otherwise indicated were purchased from Sigma-Aldrich (St. Louis, MO, USA).