A7016

Total protein from the CRC cell lines SW480, SW620, HCT116, HT29, and LoVo cells were separated by 10% SDS‐PAGE and transferred onto PVDF membranes. The following polyclonal primary antibodies were used: anti‐E‐cadherin (1:1000 dilution, AF6759, Beyotime), anti‐fibronectin (1:1000 dilution, 26836S, Cell Signaling Technology), anti‐β‐Catenin (1:1000 dilution, AF0066, Beyotime), anti‐EMCN (1:1000 dilution, PA5‐21395, Thermo Fisher Scientific), anti‐FLAG (1:1000 dilution, AF519, Beyotime), anti‐IgG (1:1000 dilution, A7016, Beyotime), anti‐β‐actin (1:4000 dilution, 20536‐1‐AP, Proteintech), and anti‐GAPDH (1:5000 dilution, 10494‐1‐AP, Proteintech). HRP‐conjugated anti‐rabbit IgG (1:3000, SA00001‐2, Proteintech) and HRP‐conjugated anti‐mouse IgG (1:2500 dilution, 7076S, Cell Signaling Technology) were used as secondary antibodies. The bands were visualized using an enhanced chemiluminescence system (Amersham Pharmacia Biotech).

Macrophages or adipocytes were fixed with formaldehyde at room temperature for 10 min, washed with PBS, and collected. The cells were then placed in an ultrasonic water bath (60 W, SCIENTZ-48, SCIENTZ, China). The long-stranded DNA of the gene was broken into 200- to 1,000-bp DNA fragments and centrifuged at 16,000 × g for 15 min at room temperature to obtain the supernatant. A total of 20 µL of supernatant was employed as the input. The c-Jun (ab32137, 2.5-5 µg/10⁶ cells) and STAT3 (ab76315, 2.5-5 µg/10⁶ cells) antibodies and their corresponding IgG antibodies (A7016; Beyotime, China) were added to the remaining supernatant, which was incubated overnight at 4°C. Magnetic beads were then added for 2 h at room temperature (CST #9005). After centrifugation at 16,000 × g for 2 min at 4°C, the precipitate was eluted stepwise with a low-salt buffer solution, high-salt buffer solution, and NaCl solution to remove chromatin. EDTA, Tris-HCl, and protease were added to the sample, which was then incubated at 65°C for 1 h. Finally, the phenol-chloroform method was used to extract the 50-µL purified product for PCR detection. The primer details are outlined in Supplementary Table 2.

Cytosolic extracts from the hippocampus were prepared as described in the manufacturer’s instructions in the Nuclear and Cytoplasmic Extraction Kit (CW0199S, CoWin Biosciences). Cytoplasmic protein was divided into three groups: Input, DHX9, and IgG (negative control). The Input group was temporarily stored in a −80°C freezer and the remaining two groups were subjected to non-contact ultrasonic lysis, "M" strength, 5 min, 3 times. Then 1 μL rabbit IgG and 20 μL Protein A + G Agarose beads were added and the samples were incubated at 4°C for 2 h. After blocking, the supernatant was collected. Next, rabbit anti-DHX9 antibody or normal rabbit IgG antibody (1 μg, A7016, Beyotime) was added to the corresponding samples, which were then rotated at 4°C overnight. Next, protein A + G agarose gels (P2055, Beyotime) were added and the samples were incubated with rotation for 1 h at 4°C. The samples were washed three times with RIP wash buffer. Finally, RNA was eluted with TE buffer and extracted with Trizol (R401-01, Vazyme). Reverse transcription was followed by qPCR, and U6 RNAs was used as target reference control.

The co-IP analysis was carried out as previously described [14 ]. We used antibodies against CIP2A, α-syn, or rabbit IgG (A7016, Beyotime Biotechnology, Co., Ltd., China). The agarose affinity gels used for the Co-IP were purchased from Beyotime, P2006. Briefly, the lysed SH-SY5Y cell samples were incubated with the antibodies, and then Protein A beads were added into the mixture and incubated. The beads were washed and solubilized in SDS sample buffer and then assessed by immunoblotting assays.

The third passage BMSCs were seeded in a 6-well culture plate with prepared cell sheets at a density of 1. 5 ×10⁴ cells/mL, and the cells were incubated overnight. When the cells were 50% confluent, the cell sheets were washed three times by PBS. The cells were then fixed with 4% formalin for 30 min, followed by washing three times with PBS, and blocked with 5% bovine serum albumin for 30 min. The experimental and control group cells were then incubated with anti-GARP antibody (NBP2-68740; 1:100; Novus Biologicals) and homologous anti-IgG antibody (A7016; 1:100; Beyotime Institute of Biotechnology, Haimen, China) at 4 °C in the dark overnight respectively, and the cell sheets washed three times with PBS. The cells were then incubated with FITC secondary antibody (A0562; 1:100; Beyotime Institute of Biotechnology) at room temperature for an hour. Cell sheets were washed three times with PBS. Then cells were incubated with 4′, 6-diamidine-2′-pheynylindole dihydrochloride (DAPI; Invitrogen, Carlsbad, CA, USA) for 10 min in the dark, and then washed as previously described. The cell sheets were imaged using an immunofluorescence microscope (Olympus, Tokyo, Japan).

The HCM cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail (Beyotime) and then incubated with Protein A/G Magnetic Agarose Beads (Thermo Fisher) for 2 hours at 4°C. Subsequently, the supernatants were collected after centrifugation at 1000 g for 5 minutes, followed by incubation with non‐specific IgG (1 μg, A7016; Beyotime) or HMGB1 antibody (10 μg, H9539; Sigma‐Aldrich) overnight at 4°C. After washing with PBS for three times, the protein A/G beads were detected by Western blotting as described above.

For the IP experiment, cell lysate was incubated with 50 μL of slurry anti-Flag M2 affinity gel (A2220, Sigma) to enrich Flag-tag proteins. Meanwhile, another part of cell lysate was incubated with the target antibody coupled with the protein A beads (161–4013, Bio-Rad) overnight at 4 °C to enrich AnxA6-interacting proteins [5 (link), 7 (link)]. As a negative control, the primary antibody was replaced with the normal rabbit IgG (A7016, Beyotime) to eliminate the nonspecific binding protein. The enriched proteins were subjected to isolation on SDS-PAGE to test the target protein by western blotting.
For western blotting, protein samples from cell lysates or immunoprecipitation (IP) were separated by 7.5–10% SDS-PAGE gel, and proteins were then transferred onto PVDF membranes (Millipore, Billerica, MA). Membranes were then blocked with 5% nonfat milk, incubated with the primary antibody overnight at 4 °C, and then incubated with horseradish peroxidase–linked secondary antibody for 1 h at room temperature. After adding ECL reagents, protein signals were detected with a luminescent image analyzer. The density of the target band in western blot was measured with Image J software for semi-quantification of staining signals.