Mouse anti n myc

Western blotting was performed as previously described [21]. The primary antibodies used were as follows: rabbit anti‐DDX21 (1 : 500; Catalogue number A300‐629A; Bethyl Laboratories, Montgomery, TX, USA), rabbit anti‐CEP55 (1 : 500; Catalogue number 81693S; Cell Signaling Technology, Danvers, MA, USA) or mouse anti‐N‐Myc (1 : 1000; Catalogue number sc‐53993; Santa Cruz Biotechnology). The secondary antibodies used were as follows: goat anti‐rabbit (Catalogue number 12‐348) or goat anti‐mouse (Catalogue number 12‐349) (1 : 10 000; both from MERCK, Burlington, MA, USA). The anti‐actin antibody (1 : 15 000; Catalogue number A3853; Sigma) was used as loading control.

Whole-cell lysates, nuclear extracts, or acid extracts were prepared from cells pelleted after washing with phosphate-buffered saline (PBS). Samples were diluted in NuPAGE LDS Sample Buffer (Novex) and 10% 2-mercaptoethanol. Protein samples were run on NuPAGE Bis-Tris mini gels (Novex), and membrane transfer was performed in 1× MES NuPAGE buffer (Novex). The following antibodies were used for probing of membranes: Primary antibodies–mouse anti-c-Myc (Santa Cruz Biotechnology, #sc-40), mouse anti-n-Myc (Santa Cruz Biotechnology, #sc-53993), mouse anti-H3 (Upstate/Millipore Sigma, #05-499), rabbit anti-H3.3 (Millipore Sigma, #09-838), rabbit anti-H4 (Millipore Sigma, #04-858), rabbit anti-H3K27M (Millipore Sigma, #ABE419), mouse anti-Cas9 (Cell Signaling, #14697), rabbit anti-H3K27me3 (Cell Signaling, #C36B11), rabbit anti-H3.3S31p (AbCam #ab92628), rabbit anti-H3K27ac (Abcam, #ab4729), rabbit anti-K36me3 (Abcam, #ab9050), rabbit anti-NOTCH (Abcam, #ab52627), rabbit anti-ASCL1(MASH1) (Abcam, #ab74065), mouse anti-beta actin (Sigma, #A1978), rabbit anti-RBPJ (Millipore-Sigma #ABE384); secondary antibodies—IRDye 680RD goat anti-mouse (1:10,000; LiCor) and IRDye800CW goat anti-rabbit (1:10,000; LiCor). Blots were imaged, data were captured on a Licor Odyssey CLx, and quantification was performed with the Licor ImageStudio software.

To measure ATM protein expression, nuclear lysates were isolated with an NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo) with protease and phosphatase inhibitor (Thermo Fisher) included. Total protein lysates were extracted by RIPA buffer (Sigma). Protein concentrations were measured using the Bradford reagent (Bio-rad). Equal amounts of protein lysate were loaded onto a 4–15% SDS/PAGE pre-cast TGX Stain-Free Gel (Bio-Rad) and subsequently transferred onto PVDF membrane (Bio-Rad) at 30 V at 4 °C overnight. Immunoblot analysis was performed with the following antibodies: rabbit-anti-ATM (Novus, NB#100–104), mouse-anti-N-MYC (Santa Cruz, #SC-53993), rabbit-anti-NSE (Millipore, # 2716128), rabbit-anti-PSA (Dako, # 20035991), mouse-anti-CgA (Millipore, # MAB5268), rabbit-anti-AR (Millipore, #06–680), mouse-anti-GAPDH (Abcam, #ab9484), rabbit-anti-p84 (Invitrogen, #PA5–27816), rabbit-anti-EZH2 (Cell Signaling Technology, #5246). Following incubation with the appropriate secondary horseradish antibodies (Bio-Rad), blots were developed using the Chemiluminescent Substrate kit (Thermo).