His tag

The 10-week-old ovaries protein of wt and tg mice were lysed by cold RIPA buffer (Beyotime) for 30 min on ice, added to 5× SDS-PAGE loading buffer (Beyotime), and boiled at 100 °C for 10 min. Then, the lysates were separated by 8-12% SDS-PAGE and transferred electrophoretically onto PVDF membrane (Millipore, USA). After being blocked with 5% defatted milk powder in TBS-T buffer (20 mM Tris/HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20), PVDF membranes were incubated with the primary antibodies (His-tag, Proteintech, Wuhan, China). The expression level of the Pai protein was marked by that of His-tag. After incubation with the HRP-conjugated secondary antibody (Santa Cruz, USA), the signals were measured using ECL reagents (Vazyme) using the Chemiluminescent Imaging System (Tanon, China). GAPDH (Proteintech) was used as an endogenous loading control.

The cells that grew on the slides were fixed, permeabilized, blocked and incubated with MYC (1:100) (Abcam), p-ANXA2 (Tyr23, 1:100) (Santa Cruz) or His-tag (1:100) (Proteintech) at 4 °C overnight. The bound primary antibodies were detected using goat anti-mouse IgG-FITC or goat anti-rabbit IgG H&L (1:100) (Abcam) at 37 °C for 1 h. The fluorescence was detected via confocal microscopy (General Electric Company, Fairfield, CT, USA).

Total protein was isolated using RIPA buffer (Applygen, Beijing, China) with protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). Nuclear and cytoplasmic protein was isolated using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Immunoblotting was performed with primary antibodies against ANXA2 (1:200), p-ANXA2 (Tyr23, 1:200), VEGF (1:200) (Santa Cruz Biotechnology, Dallas, Texas, USA), MYC (1:1000), p-SRC (Tyr418, 1:1000) (Abcam, Cambridge, UK), Ubiquitin (1:500), Histone H3 (1:1000) (CST, Danvers, MA, USA), HIF1A (1:500), SRC (1:500), HA-tag (1:3000), His-tag (1:3000) (Proteintech, Wuhan, China). GAPDH (1:500) (Proteintech) was used as a loading control. Secondary antibodies (Goat anti-Mouse IgG and Goat anti-Rabbit IgG, 1:5000) were purchased from Applygen. The signals were visualized with a super enhanced chemiluminescence (ECL) detection reagent (Applygen). Quantitative analysis of immunoblotting was performed using ImageJ (Ver. 1.52a, NIH image, Bethesda, MD, USA).

The primary antibodies were used as follows: GAPDH (Proteintech, 60004-1-Ig), CapZβ (Proteintech, 25043-1-AP), ARPC1B (Sigma-Aldrich, HPA004832), p16-Arc (Abcam, ab51243), N-WASP (Novus Biologicals, NBP1-82512), His-tag (ProteinTech, 66005-1-Ig), β-actin (ProteinTech, 60008-1-Ig), α-tublin (ProteinTech, 66031-1-Ig), VPS8 (ProteinTech, 15079-1-AP), λ-tublin (Sigma-Aldrich, T6557), HSC70 (Santa Cruz, sc-24), VPS34 (CST, 4263), CapZa1/a2 (Developmental Studies Hybridoma Bank, mAb 5B12.3), anti-pan actin (Cytoskeleton, AAN02-s), RAB5A (CST, 3547 or 46449), Calnexin (ProteinTech, 10427-2-AP), LAMP1 (CST, 9091), TGN46 (Bio-Rad, AHP500GT), EEA1 (CST, 3288), MYC-tag (ProteinTech, 67447-1-Ig), Flag-tag (Sigma-Aldrich, F3165), HA-tag (Sigma-Aldrich,11867423001), StrepMAB-Classic HRP conjugate (IBA, 2-1509-001), Rabex-5 (Santa Cruz, sc-166049), Rabaptin-5 (ProteinTech, 14350-1-AP), EGFR (Santa Cruz, sc-373746), goat anti-mouse IgG (H+L) secondary antibody (Invitrogen, 31430), goat anti-rabbit IgG (H+L) secondary antibody (Invitrogen, 31460), and goat anti-rat IgG (H+L) secondary antibody (Invitrogen, 31470).

Cells plated on slides were transfected with UHRF1-His and c-Jun-Flag for 48 h. Then, the cells were fixed in 4% paraformaldehyde at room temperature (RT) for 12 min and permeabilized in 0.5% TritionX-100 (PBS) for 5 min. The slides were blocked with 3% BSA (PBS) for 1 h at RT. After that, the cells were incubated with primary antibodies, Flag-tag (Proteintech) and His-tag (Proteintech), at RT for 2 h, followed by Alexa Fluor 488-conjugated and 546-conjugated secondary antibodies (Invitrogen). The cells counterstained with DAPI (1 μg/mL; Sigma) were visualized using a confocal microscope (Olympus).

Bacterial strains, plasmids, and oligonucleotides used in this study are listed in Supplementary Table 3. E. coli DH5α strain was used for cloning and E. coli BL21(DE3) for protein production and purification. P. luminescens subsp. laumondii TTO1 DNA was used as the template for cloning. Bacteria were grown in Lysogeny Broth (LB), with agitation at 37 °C. When required, media were supplemented with ampicillin (100 μg mL⁻¹), streptomycin (100 μg mL⁻¹) or kanamycin (50 μg mL⁻¹). Gene expression from pET vectors derivatives was induced by the addition 500 μM isopropyl β-D-1-thiogalactopyranoside (IPTG). SDS-PAGE, Western-blots and immunodetection have been performed using standard procedures. Gels were stained using InstantBlue^TM (Sigma-Aldrich) or transferred onto nitrocellulose membrane, and immunodetected with His-Tag (clone 1B7G5, Proteintech catalogue #66005-1-Ig, dilution 1/5,000), Strep-Tag Classic (clone Strep-tag II, Bio-Rad catalogue #MCA2489, dilution 1/1,000) or anti-FLAG (clone M2, Sigma-Aldrich catalogue #F3165, dilution 1/1,000) monoclonal antibodies, and secondary goat anti-mouse antibodies conjugated to the Alkaline Phosphatase (AffiniPure, Jackson ImmunoResearch catalogue #115-055-003, dilution 1/5,000), and revealed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) in presence of 10 mM MgCl₂ in alkaline buffer (pH 9).

Rat-derived monoclonal antibodies against His-tag, Flag-tag, HA-tag, and β-actin (Proteintech, Rosemont, IL, USA) were diluted in block buffer (1:5000) to be used for Western blot analysis. We diluted rabbit-derived polyclonal antibody against Sf-caspase-1 (also provided by Professor Nor Chejanovsky), which can recognize full-length, large subunits of Sf-caspase-1 and Sl-caspase-1, 1:1000 in block buffer to be used for western blotting. A polyclonal antibody against SlDronc, which can recognize full-length and large subunits of SlDronc, was produced using a SlDronc fragment purified in E. coli as an antigen to immunize rabbits. We produced a polyclonal antibody against SfIAP, which can also recognize full-length, cleaved SlIAP, using a SfIAP fragment purified in E. coli as an antigen to immunize rabbits.