Ab122525

Cells were lysed in RIPA Buffer (Sigma-Aldrich) and protein concentration was determined by using the BCA assay kit (Sigma-Aldrich). Total protein (40 μg) was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% or 5% gel and electrotransferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were then incubated in 5% nonfat dry milk in Tris-buffered saline (TBS, pH 7.6) and then overnight at 4 °C with a rabbit monoclonal anti-DHC2 (#ab122525; Abcam) at a dilution of 1:1000, anti-KIF2B (#ab98214; Abcam) at a dilution of 1:2000 or anti-Phospho-H2A.X (#9718; Cell Signaling Technology) at a dilution of 1:1000. Membranes were then incubated at 37 °C for 1 h with an HRP-conjugated anti-rabbit IgG antibody (#7074; Cell Signaling Technology) at a dilution of 1:2000. After three washes with TBS, membranes were briefly incubated with chemiluminescent HRP substrate (#WBKLS0100; Millipore) and were photo-developed in Image Station 2000 MM (Kodak, Rochester, Minnesota, USA). Quantity One 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA) was used to quantify the density of the target protein in the membranes.

Nude mouse xenografts (see below) and ex vivo GBM tissue samples were subjected to immunohistochemical staining of DHC2 and KIF2B proteins using a standard protocol according to a previous study40 (link). DHC2 (#ab122525; Abcam) and KIF2B (#ab98214; Abcam) antibodies were used at dilution of 1:200. The stained tissue sections were reviewed and scored separately by two pathologists blinded to the clinical parameters under a microscope. Expression level of these proteins were assessed by the percentage of positive cells and staining intensity, i.e., the percentage of positive cells was scored as follows: 0% (absent), 1–5% (sporadic), 6–25% (local), 26–50% (occasional), 51–75% (majority), and 76–100% (most). The staining intensity of cancer cells was graded as 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), and 3 (strong staining, brown). An intensity score of 2with at least 50% of positive cells was considered as high expression (or overexpression), and <50% of positive cells or <2 in intensity score was regarded as low expression.