Ranbpm

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).

Formalin-fixed, paraffin-embedded samples were sectioned at 5 μM. Sections were treated with antigen retrieval buffer. Specifically, incubation with antibodies against RanBPM (1:50 dilution; Santa Cruz, USA), p21 (1:100 dilution; CST) was carried out overnight at room temperature. Slides were incubated in secondary antibody. The protein levels of RanBPM and p21 in the tumor specimens from NSCLC patients were reviewed and scored under a light microscope. Each specimen was quantified and assigned a score based on the intensity of the membrane, cytoplasmic, and/or nucleic staining by a visual grading system (0–3) (grade 0, no staining; grade 1, weak staining; grade 2, moderate staining, grade 3, strong staining) and the extent of stained cells (0% =0, 1–24% =1, 25–49% =2, 50–74%=3, 75–100%=4). The final immunoreactive score ranges from 0 (no staining) to 12 (strong staining).