Anti cdk2

Human glioblastoma cells or tumor tissues were harvested and washed with ice-cold PBS. Then, samples were suspended in SDS sample buffer, boiled for 10 min, and centrifuged at 12,000 rpm for 15 min. Thirty micrograms of protein samples were separated using 8–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore Corporation). The PVDF membrane was blocked with 5% milk for 1 h and then incubated with a primary antibody at 4°C overnight. Subsequently, the PVDF membrane was probed with the secondary antibody and visualized by enhanced chemiluminescence (ECL) (Beyotime Institute of Biotechnology, Haimen, China). The primary antibodies were as follows: anti-G9a (1:500, Santa Cruz), anti-a-tubulin (1:2000, Sigma), anti-CDK1 (1:1000, Abcam Cambridge, MA, United States), anti-CDK2 (1:500, Santa Cruz), anti-Cyclin A2 (1:2000, Abcam), anti-Cyclin B1 (1:3000, Abcam), anti-MMP1 (1:1000, Abcam), anti-N-cadherin (1:5000, Abcam), anti-LC3B (1:1000, Cell Signaling Technologies, Danvers, MA, United States), anti-p62 (1:1000, Abcam), anti-c-Myc (1:1000, Abcam), and anti-H3K9me2 (1:2000, Abcam). Horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000), goat anti-rabbit IgG (1:20,000), and rabbit anti-goat IgG (1:10000) (KPL, Gaithersburg, Maryland, United States) were used as secondary antibodies.

The following agents were obtained from commercial sources: vascular endothelial growth factor‐A 165 (Merck Millipore, Billerica, MA, USA); anti‐phospho‐VEGFR‐2 (Y951) (Abcam, Cambridge, UK); anti‐phospho‐p70^S6K (T421/S424), anti‐phospho‐Akt (S473), anti‐phospho‐ERK (T202/Y204), anti‐phospho‐p38^MAPK (T180/Y182), anti‐phospho‐pRb (S780) and anti‐phospho‐pRb (S807/S811) (Cell Signaling, Beverly, MA, USA); anti‐phospho‐tyrosine (BD Biosciences, Bedford, MA, USA); anti‐VEGFR‐2, anti‐vascular endothelial (VE)‐cadherin, anti‐integrin β1, anti‐ILK, anti‐p70^S6K, anti‐Akt, anti‐ERK, anti‐p38^MAPK, anti‐Cdk2, anti‐Cdk4, anti‐cyclin D, anti‐cyclin E, anti‐actin antibodies and mouse and rabbit IgG‐horseradish peroxidase conjugates (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Alexa Fluor 488–conjugated goat anti‐mouse IgG (Life Technologies, Grand Island, NY, USA).

Cells and exosomes were lysed using CHAPS lysis buffer (30 mM Tris-Cl, pH 7.5; 150 mM NaCl; 1% CHAPS) mixed with 25X protease inhibitor (Roche), and sonication. Proteins were quantified by Bradford method (Bio-Rad). Electrophoresis was performed using 10% acrylamide gel, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) and probed with the following antibodies: anti-TSG101 (Abcam), anti-HSC70, anti-GAPDH, anti-Calnexin, anti-C/EBPβ, anti-CDK2 (all from Santa Cruz Biotechnology), anti-FOXO-1 (Cell Signaling), and appropriate species-specific HRP-conjugated secondary antibodies (Santa Cruz), and detected using ECL reagent (Roche).

Lysates (40 μg) of freshly isolated or cultured cells were analyzed with anti-p21 (Santa Cruz) anti-phospho ERK antibodies (Cell Signaling); anti-ERK (Cell Signaling), anti-CDK2 (Santa Cruz) and anti-β-actin (Sigma) served as loading controls. Macrophage whole cell lysates (20 μg) were probed with anti-phospho-STAT1 (Cell Signalling) and anti-iNOS (Abcam).

Kidney tissues and cells were lysed in mammalian protein extraction buffer (GE Healthcare, Milwaukee, WI, USA) containing a protease and phosphatase inhibitor cocktail (GenDEPOT Inc., Katy, TX, USA). Approximately 20–50 µg of the lysates were electrophoresed and transferred to polyvinylidene difluoride membranes (PVDF, Millipore, Billerica, MA, USA). Membranes were blocked with 3% nonfat milk and incubated with primary antibodies overnight at 4 °C. The following antibodies were used at the indicated dilutions: anti-cyclin A, anti-cyclin D1, anti-CDK2, anti-CDK4, anti-p27^Kip1, anti-KLF5, anti-β-actin (1:500; Santa Cruz Biotechnology), anti-p-Erk, anti-Erk, anti-p-JNK, anti-JNK, anti-p-p38, anti-p38, and Egr1 (1:1000; Cell Signaling Technology). After washes, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology) for 1 h at room temperature. Immunoreactive signals were detected using an ECL detection system (Millipore).