Ab32072

The transfected cells were lysed in lysate buffer (mixture of 50 mmol/L Tris ‐ HCl (pH 7.4), 150 mmol/L NaCl, 10% glycerol, 1 mmol/L EDTA, 0.5% NP‐40 and protease inhibitor) and centrifuged to move cell debris. Cleared cell lysate was incubated with 1 μg anti‐HA (ab9110, 1:70, Abcam, Cambridge, UK), myc (ab32072, 1:100, Abcam, Cambridge, UK), USP7 (ab109109, 1:1000, Abcam, Cambridge, UK), KDM6B (ab38113, 1:100, Abcam, Cambridge, UK) or anti‐FLAG antibody (ab205606, 1:1000, Abcam, Cambridge, UK) and 15 μL protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hours. After extensive washing, the beads were boiled at 100℃ for 5 minutes. Proteins were resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, Temecula, CA, USA), followed by immunoblotting. To detect endogenous protein interactions, cells were lysed in ice‐cold lysis buffer. Cleared cell lysates were incubated with 5 μg anti‐USP7 antibody (ab4080, 1:1000, Abcam, Cambridge, UK) and 20 μL protein A/G beads at 4°C overnight. The anti‐USP7 antibody was used to detect the endogenous KDM6B.

Immunohistochemical staining was performed as previously described [20 (link)]. The negative control was detected with the primary antibody replaced by PBS. Immunohistochemical staining was scored by two independent observers who were blinded to the patients’ clinical data. The scoring system for grading the level of YAP was reported previously [22 (link)]. The expression level was evaluated by immunohistochemistry (IHC) score calculated by multiplying a proportion score and intensity score. The proportion score reflected the fraction of positive-stained cells (0, none; 1, ≤ 10%; 2, 10–25%; 3, 25–50%; and 4, > 50%), and the intensity score revealed the staining intensity (0, no staining; 1, weak; 2, intermediate; and 3, strong). Finally, the total score was calculated. High and low protein expression levels were defined using the mean score of all samples as a cutoff point. With these criteria, tissue staining could be interpreted as “low” or “high.” The primary antibodies used were anti-YAP (Proteintech Group, 13584-1-AP, IL, USA), anti-Ki67 (Proteintech Group, 27309-1-AP), and anti-c-myc (Abcam, ab32072, Cambridge, UK). For hematoxylin-eosin (H&E) staining, fresh subcutaneous tumors isolated from mice were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin for histological examinations. Sections with thickness of 4 μm were cut and stained with H&E.

The tissues and cells were lysed in the radio‐immunoprecipitation assay lysis buffer (Sigma–Aldrich) to collect total protein. The concentration of the isolated protein was examined using the bicinchoninic acid method. Next, an equal amount of protein sample (40 μg each lane) was separated by 10% SDS‐PAGE and loaded onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk at 4˚C overnight, and incubated with the primary antibodies against TGIF2 (1:1,000, 11522‐1‐AP; Proteintech Group, Inc., Wuhan, Hubei, China), β‐catenin (1:1,000, #13‐8400; Thermo Fisher Scientific), Wnt1 (1:1,000, #36‐5800; Thermo Fisher Scientific), c‐Myc (1:1,000, ab32072; Abcam Inc., Cambridge, MA, USA), c‐FOS (1:1,000, ab222699, Abcam), Survivin (#PA1‐16836, Thermo Fisher Scientific), and GAPDH (1:10,000; ab8245; Abcam) at 4℃ overnight. Thereafter, the membranes were incubated with HRP‐labeled goat anti‐rabbit IgG (1:5,000; ab6721; Abcam) or goat anti‐mouse IgG (1:2,000; ab6789; Abcam) at room temperature for 1.5 h. The blot bands were developed using the enhanced chemiluminescence system (Thermo Fisher Scientific) and analyzed using Image J software (Version 1.46; NIH, Bethesda, MD, USA).

We chose the most representative genes with a positive or negative correlation with the prognosis of GBM patients as the research objects, whose IHC staining was downloaded from the Human Protein Atlas (HPA: http://www.proteinatlas.org/) and analyzed. This allowed us to assess differences in cell senescence-associated genes expression at the protein level.
We collected section from paraffin-embedded tissues of human glioma and peritumor. We dewaxed and dissociated the sections and rehydrated sections. After heating in tris-EDTA buffer, we blocked slides using 5% gout serum and incubated slides with primary antibody (PTTG1, 1:500, #ab128040; Abcam) (MYC, 1:100, #ab32072; Abcam) at 4 °C overnight. Then the slides were incubated with secondary antibody and the images were captured using a Leica DM 2500 microscope.