Ab109307

Tissues were fixed using 4% formalin for 48 h. Then, the tissues were embedded using OCT (Sigma, USA). The tissues were sectioned in 8 μm thickness. After antigen repair, 3% H₂O₂ treatment, washing with PBS, and blocking with 5% goat serum. The primary antibodies (Rabbit monoclonal to c-myc, 1:1500, #ab32072, Abcam; Rabbit monoclonal to axin, 1:1000, #ab109307, Abcam; Rabbit monoclonal to beta catenin, 1:1000, #ab32572, Abcam) were used to cultivate tissues at 4 °C overnight. After washing, the tissues were cultured with second antibody (Goat anti-rabbit lgG, 1:2000, #ab205718, Abcam) for 2 h. DAB reagent (#34002, Thermo Fisher, USA) was used to treat tissues. The sections were visualized and captured using an inverted optical microscope (Olympus Corporation). The expression intensity was analyzed through Image J software.

Axin2 validation was conducted using both human RKO cells and intestinal sections of APC^loxp/loxp vs APC^fl/fl; ROSA26^CreERT2. Briefly, RKO cells were GSK3-β (CHIR)-treated for 48 hr vs controls, harvested, 4% paraformaldehyde fixed for 10 min and subsequently washed and permeabilized with 0.25% Triton X-100. Cells were then blocked with 2.5% horse serum and stained for Axin2 Abcam (ab109307, EPR2005, 1:100) for flow cytometry. Intestinal epithelial sections were stained with Axin2 (ab109307, EPR2005, 1:25, AR; Citrate pH = 6).

The collected tissues and treated cells were lysed in RIPA buffer (Abcam, MA, USA) on ice for 40 min to extract total protein. After being quantified with BCA kit (ab102536; Abcam, MA, USA), 50 μg of total protein was separated by 12% SDS-PAGE gels and electrophoretically transferred onto PVDF membranes, which were subsequently blocked in skimmed milk (8%) for 2 h. The membranes were incubated overnight at 4 ℃ with primary antibodies against Bax (ab32503; Abcam, MA, USA), Bcl-2 (ab32124; Abcam, MA, USA), CRIM1 (PA5-34,410; Thermo Scientific, MA, USA), ERK1/2 (ab17942; Abcam, MA, USA), T202/Y204 phosphorylated-ERK1/2 (p-ERK1/2) (ab214362; Abcam, MA, USA), Axin2 (ab109307; Abcam, MA, USA), β-catenin (ab32572; Abcam, MA, USA), and β-actin (ab8226; Abcam, MA, USA), followed by rinsed thrice with NaCl/Tris (TBS) supplemented with 0.1% Tween 20 (TBS-T) before the 2 h incubation of secondary antibody of Goat Anti-Rabbit IgG H&L (HRP) (ab6721; Abcam, MA, USA). Finally, bands were visualized by ECL Substrate Kit (Abcam, MA, USA), and quantified using Image J software (USA).

60S ribosomes were a generous gift from the Puglisi lab, and were also purified in‐house as described previously (Johnson et al, 2019 (link); Lapointe et al, 2021 (link)).For reconstituting 60S Ribosome UFMylation, approximately 50 nM purified 60S ribosomes were mixed with, 0.5 μM UBA5, 1 μM UFC1, 0.5 μM UFM1, and 100 nM UFL1/UFBP1 in a reaction buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂ and 5 mM ATP and incubated at 30°C for 10 min or indicated time duration. The reaction was stopped by the addition of SDS loading buffer (1× final) and run on a 4–12% SDS–PAGE gel under reducing conditions followed by immunoblotting using indicated antibodies.
Ribosome UFMylation assay as described in Fig 4E was performed at 30°C with 1 μM UFC1, 0.5 μM UBA5, 0.5 μM UFM1, 0 nM or 75 nM UFL1/UFBP1, 50 nM purified 60S ribosomes, and increasing concentrations of CDK5RAP3 (0, 38, 75, 150 or 375 nM) in a reaction buffer of 25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂ and 5 mM ATP. The reaction was quenched by the addition of SDS‐Load buffer (1× final) with reducing agent. Western blots show 10 min reaction time; immunoblots for RPL26 (Abcam, 59567) and UFL1 (Bethyl, A303‐455M) were run on the same gel, which was cut and probed for these proteins separately. Immunoblots for CDK5RAP3 (Bethyl, A300‐871A) and UFM1 (Abcam, Ab109307) were run on separate gels.

For immunofluorescence staining, the slides were incubated with antibodies against CACNA1C (ACC‐003; Alomone Labs; 1:200), E‐cadherin (610182; BD Biosciences; 1:200), ZO‐1 (ab221547; Abcam; 1:200), vimentin (5714; CST; 1:200), human leukocyte antigen (HLA) (ab70328; Abcam; 1:200), CK14 (ab7800; Abcam; 1:200), and P‐cadherin (ab19350; Abcam; 1:200) at 4°C overnight. Subsequently, secondary antibodies (Invitrogen; 1:200) in PBT for 2 h at room temperature, washed again with PBT, and counterstained with TO‐PRO‐3 (T3605; Thermo Fisher Scientific; 1:1000) in TDW for 15 min. For Phalloidin staining, slides were incubated with Alexa Fluor 488 Phalloidin (A12379; Thermo Fisher Scientific; 1:400). For colorimetric immunohistochemistry of tissues, slides were incubated with antibodies against Ki67 (ab16667; Abcam; 1:200), β‐catenin (sc‐7963; Santa Cruz; 1:100), Axin2 (ab109307; Abcam; 1:200). Subsequently, slides were processed with secondary antibody kit (D03‐110; GBI Labs) and DAB staining kit (C09‐12; GBI Labs) following manufacturer's protocol. All the images were taken by inverted Laser Confocal Microscope (DMi8; Leica).

The cells were washed three times with PBS and lysed with RIPA buffer supplemented with protease and phosphatase inhibitors for 30 min at 4 C. For the Western blot analysis, 15 μL of the sample was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electro-transferred onto nitrocellulose membranes. The primary antibodies used were rabbit polyclonal anti-GCN5 (1:1000, ab231075, Abcam, China); rabbit monoclonal anti-Axin-2 (1:1000, ab109307, Abcam, China); rabbit monoclonal anti-β-catenin (1:1000, ab32572, Abcam, China) and mouse monoclonal phosphorylated β-catenin (1:500, 05-665-AF647, Millipore, United States); rabbit monoclonal anti-H3K9ac (1:1000, ab10812, Abcam, China); rabbit monoclonal anti-RUNX2 (1:1000, ab192256, Abcam, China); rabbit monoclonal anti-OCN (1:1000, ab102936, Abcam, China); and rabbit monoclonal anti-OPN (1:1000, ab63856, Abcam, China). For normalization of protein loading, GAPDH antibody (1:1000, ab245357, Abcam, China) was used. The protein bands were measured with ImageJ 1.48v software and normalized to the corresponding GAPDH bands. The relative density of each target protein normalized to the control was used to represent the changes in expression of target proteins.