Anti tp53

Total protein was extracted from tissues and cell pellet lysis, and the concentration of protein in the lysate was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, United States). Proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore; Merck KGaA, Darmstadt, Germany). After blocking with 5% skimmed milk for 1 h, the membranes were probed with primary antibodies at 4°C overnight: anti-TP53 (1:1000) and LC3 (1:500) were purchased from Cell Signaling Technology (CST; Danvers, MA, United States), while anti-p-AKT^Ser473 (1:500), anti-p-mTOR (1:500), anti-p-P70S6K, anti-p-4EBP1, and P62 (1:500) were purchased from Sigma (St. Louis, MO, United States), and anti-GAPDH (1:5000) was purchased from CST, which served as a loading control. Then, the membrane was incubated with a secondary antibody for 2 h. The protein expression was visualized using enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific), and the concentration of proteins was quantified by Image J software.

Flow cytometry was used to measure the apoptotic cells using Annexin V as previously described.[10 (link)] Intracellular flow cytometry to measure TP53 has been described previously.[12 (link)] The following antibodies were used: anti-CD71 (FITC, 11-0719-42, ebioscience), anti-glycophorin A (PE, 12-9987-80, ebioscience), anti-TP53 (Alexa647, #2533S, cell signaling) and Annexin V (556421, BD Pharmingen).

Histopathological analysis was performed by using hematoxylin and eosin (H&E) staining. Immunohistochemical analysis of HER2 was performed to evaluate cell proliferation and downstream intracellular signaling in neoplastic tissue. Primary antibodies used in this study were anti-HER2 (1:200, CST#4290, Cell Signaling Technology, Danvers, MA, USA), anti-MUC1 (1:100, ab15481, Abcam, Cambridge, MA, USA), anti-MUC2 (1:200, sc-15334, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MUC5 (1:100, sc-21701, Santa Cruz Biotechnology), anti-Ki67 (1:100, ab16667, Abcam), anti-CyclinD1 (1:25, CST#2978, Cell Signaling Technology), anti-phospho-p44/p42 (1:100, CST#4376, Cell Signaling Technology), anti-SOX-9 (1:100, sc-20095, Santa Cruz Biotechnology), anti-TP53 (1:200, CST#2524, Cell Signaling Technology), and anti-CK19 (1:50, sc-376126, Santa Cruz Biotechnology).

RQ1 RNase‐free DNaseI (Promega, #M6101), SuperScript III First strand‐synthesis system (Thermo Fisher Scientific, #18080051), RNase A (Merck, #10109242001), anti‐EGFP (Abcam, UK, #ab290), anti‐SRSF3 (Merck, #WH0006428M8), anti‐CDKN1A (Cell Signalling Technologies, #2947), anti‐GAPDH (Cell Signalling Technologies, #2118S), anti‐TP53 (Cell Signalling Technologies, #2524), anti‐HA tag (ThermoFisher Scientific, #MA5‐25644), TaqMan® MicroRNA Reverse Transcription Kit (ThermoFisher Scientific, #4366596), SensiFAST Probe Hi‐ROX Kit (Bioline, #BIO‐82005), Pierce Firefly Luciferase Glow Assay Kit (ThermoFisher Scientific, #16176), Dynabeads Protein G beads (Thermo Fisher Scientific, #10009D), TRI Reagent (Sigma‐Aldrich, #T9424), Luminaris HiGreen qPCR Master Mix‐low ROX (ThermoFisher Scientific, #K0974), predesigned TaqMan probes (ThermoFisher Scientific, #4427975). NuPAGE 4–12% gradient Bis‐Tris gels (ThermoFisher Scientific, #NP0322BOX), ECL Western Blotting Detection Reagents (MERCK, #GERPN2209), Silencer Select predesigned siRNA (ThermoFisher Scientific, #4392420, SRSF3 assay ID s12732 and Negative Control No. 1), 2‐methylnicotinic acid imidazolide (MERCK, #913839), eBioscience™ Cell Proliferation Dye eFluor™ 670 (ThermoFisher Scientific, #65‐0840‐85) and cOmplete™ Mini EDTA‐free (Merck, #11836170001).

The dorsal root ganglion tissues were soaked in 4% formalin overnight. 5 μm paraffin sections of dorsal root ganglion was deparaffinized using xylene and rehydrated using a gradient of ethanol. Endogenous peroxidase was suppressed using H₂O₂ (3%) for 0.5 h. Then, the slices were incubated with normal goat serum (10%) and anti-IL-6 (1 : 200, Cell Signaling, USA) or anti-TP53 (1 : 200, Cell Signaling, USA) or anti-MAPK1 (1 : 200, Cell Signaling, USA) primary antibodies at 4°C overnight. The slices were washed twice in PBS and incubated with a goat anti-rabbit antibody (1 : 200) at room temperature for 60 min. Subsequently, slices were visualized using a DAB reagent. Finally, we used light microscopy to obtain immunohistochemistry images.

Antibodies used were directed against anti-H3 (Abcam, #ab1791), anti-H3.3 (Abcam, #ab176840), anti-H3K9me3 (Abcam, ab8898), anti-H3K36me3 (Abcam, #ab9050), anti-γH2A.X/phospho-histone H2A.X (Ser139) clone JBW301 (Merck Millipore, #05-636), anti-ATRX (Santa Cruz Biotechnologies, #sc15408), anti- KDM4B (Abcam, #ab191434) anti-IDH1 (Sigma Aldrich, SAB4100064), anti-IDH1^R132H (Sigma Aldrich, SAB4200548), anti-HP1α (Merck Millipore, #MAB3584), anti-PML (Merck Millipore, #MAB3738), anti-TP53 (Cell Signaling Technologies, #cst-2524), anti-Flag (Sigma, #F1804), anti-TERF1 (Alpha Diagnostics, #TRF12-S) and anti-BrdU (Abcam, #ab6326), Anti-β Actin (AC-15) (Santa Cruz Biotechnology, #sc69879), Goat anti Rabbit IgG, HRP conjugate (Merck Millipore, #AP187P), Donkey anti-Mouse IgG HRP conjugate (Merck Millipore, #AP192P), Donkey anti-Mouse IgG (H + L) Alexa Fluor 594 (Invitrogen, #A-21203), Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 (Invitrogen, #A-21206).