Ab38113

Radio immunoprecipitation assay lysis buffer (C0481, Sigma-Aldrich) was used for total protein extraction.²³ (link) The protein sample was quantified using a bicinchoninic acid kit (Beyotime, Shanghai, P.R. China), separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electrotransferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Immunoblots were incubated with primary antibodies overnight at 4°C and then incubated with horseradish peroxidase-conjugated secondary antibodies (ab99702, Abcam, Cambridge, UK). Immunoreactive bands were visualized by enhanced chemiluminescence (Baomanbio, Shanghai, P.R. China), and protein expression was normalized against β-actin. Antibodies against the following proteins were used (all from Abcam): β-actin (ab115777), FOXO6 (ab48730), c-CASP3 (ab13847), USP (ab4080), JMJD3 (ab38113), HA (ab9110), FLAG (ab1162), and Myc (ab32072). The levels being reported were relative to the mean expression of three non-cancerous samples (three adjacent non-tumorous esophageal tissues or normal esophageal cells) for each experiment.

Immunofluorescence analysis of NICD and JMJD3 was performed on CRC cell lines using the mouse monoclonal to Notch1 antibody (Abcam, # ab44986) and rabbit polyclonal to JMJD3 antibody (Abcam, #ab38113). In brief, formaldehyde fixed cells (2% formaldehyde for 10 minutes) were washed and incubated with primary antibodies for 20 minutes, then cells were washed and incubated with fluorescent secondary antibody for 15 minutes. The cover slips were mounted onto slides using the DAKO fluorescent mounting medium (Agilent, Carpinteria, CA, USA). Cells were scanned using a Leica TCS SP2 confocal microscope and Leica Confocal Software v2.7. The excitation source for Alexa 488 was a 20 mW Argon Laser at 488 nm, whereas the excitation source for Alexa 594 was a 1.2 mW Green HeNe Laser at 543 nm.

Paraffin sections of mouse tissue samples (1 day after operation) were dehydrated with gradient alcohol, treated with 3% methanol in H₂O₂ for 20 min and 0.1M PBS for 3 min (C-0005; Haoran Biotechnology, Shanghai, P.R. China) at room temperature for 20 min, followed by the addition of primary rabbit anti-human KDM6B (5 μg/mL, ab38113; Abcam, Cambridge, UK) dropwise, incubated overnight at 4°C, and washed three times with 0.1M PBS (5 min/time). The sections were then added with secondary goat anti-rabbit IgG (ab6785, 1:1,000; Abcam) dropwise and underwent further incubation at 37°C for 20 min and three washes with PBS (5 min/time), followed by incubation with horseradish peroxidase (HRP)-labeled streptavidin protein working solution (0343-10000U; Imunbio, Beijing, P.R. China) at 37°C for 20 min. After being washed three times with 0.1M PBS (5 min/time), the sections were developed using diaminobenzidine (DAB; ST033; Whiga Technology, Guangzhou, Guangdong, P.R. China), counterstained by hematoxylin (PT001; Shanghai Bogoo Biotechnology, Shanghai, P.R. China) for 1 min, returned to blue using 1% ammonia water, followed by dehydration, clearing, and finally mounting using neutral resin. The sections were then observed under a microscope, and the images in the hippocampal CA1 and CA3 regions were captured.