Ab204448

For immunofluorescence assays, dissected mosquito ovaries were dissected into phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min at RT. To block non-specific binding, ovaries were incubated for 2 h at RT with 3% bovine serum albumen in PBS with 0.1% Tween 20. Following blocking, Cas9 protein was detected using rabbit anti-Cas9 polyclonal antibody (Abcam ab204448) diluted 1:500 in PBS with 0.1% Tween 20 and incubated for 1 h at RT or overnight at 4 °C. After incubation, samples were washed three times with 0.1% Tween 20 in PBS, and the primary antibody labeled with 1:500 anti-rabbit Alexa Fluor 594 secondary antibody (Abcam ab150076). After three additional washes, ovaries were air-dried and slide mounted with ProLong Gold Antifade Reagent (Molecular Probes) and visualized using epifluorescent microscopy.

Western blotting analyses were performed as described previously^{67 (link)}. Briefly, HEK293T cells were grown in 6-well plates and transfected with 1000 ng of A3-CBE plasmids (A3A-, A3B-, A3Bctd-, A3G and A3Gctd-CBEs) and 1000 ng of EMX1-1 sgRNA plasmid with or without Ade plasmids (Ade1–4). Seventy-two hours later, the transfected cells were lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). The whole-cell lysate was used for immunoblotting. An antibody against Cas9 (1:1500; ab204448, Abcam) was used as a primary antibody, while an anti-tubulin antibody (1:2000; 10094‐1‐AP, Wuhan Sanying) was used as the loading control.

The GBM cell lines A172 and U251 were purchased from the Institute of Fudan IBS Cell Center (HNC241, HNC1088, FDCC, Shanghai, China), and the human glioblastoma LN229 cell line was kindly provided by Guoxiang Jin (the First Affiliated Hospital, Army Medical University). Normal human astrocytes (NHA) were bought from the KeyGEN Biotech Company (KG578, Nanjing, China). All cells were cultured at 37 °C in 5% CO₂ in Dulbecco’s modified Eagle medium (DMEM, HyClone) containing 10% (v/v) fetal bovine serum, 4 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 1% nonessential amino acids (Thermo, Carlsbad, CA, USA). Antibodies against Cas9 (Abcam, ab204448), Nanos3 (Abcam, ab70001), cyclin D1 (Abcam, ab40754), Gapdh (Abcam, ab37168) and α-tubulin (Abcam, ab7291) were purchased from Abcam (Cambridge, UK). The Cell Counting Kit-8 (CCK-8) reagent (CK04) was purchased from DOJINDO Molecular Technologies, Inc. (Japan). An Alkaline Phosphatase Stain Kit (SK-5300) was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Puromycin dihydrochloride (60210ES25) was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China). Blasticidin S hydrochloride (15205), Doxorubicin hydrochloride (DOX) (D1515), DMH2 (SML 1535), and BMP4 (B2680) were purchased from Sigma Aldrich (St. Louis, MO, USA), and temozolomide (TMZ) was purchased from Merck (T2577, Darmstadt, Germany).