Ma1 91878

HeLa cells were transfected with wild‐type Flag‐PBRM1 or Flag‐PBRM1–3m and split into eight‐well chamber slides 1 day after transfection. The next day, the cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X‐100 for 15 min, followed by three washes with PBS. The cells were blocked with 5% bovine serum albumin for 60 min and incubated with primary anti‐Flag antibody (Invitrogen, Catalog #MA1‐91878) overnight at 4 °C. The next day, the cells were washed three times with PBS and incubated with fluorescent secondary antibodies (Santa Cruz Biotechnology, sc‐2010) for 1 hr. Nuclei were stained using DAPI (Invitrogen, Catalog #D1306). Labeled cells were observed using a green (488 nm) laser under a confocal microscope (NikonC1 Plus laser; Nikon, Melville, NY, USA) to detect PBRM1 subcellular localization.

Cellular proteins were extracted using RIPA lysis buffer (G2002, Servicebio, Wuhan, China), and protein concentrations were determined using a BCA kit (G2026, Servicebio, Wuhan, China). Subsequently, proteins were separated through electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore, Burlington, Massachusetts, USA). After blocking with 5% non-fat milk, the membranes were incubated with specific primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies for 1 h at 25 °C. Chemiluminescence reagent (G2014, Servicebio, Wuhan, China) was employed for signal detection using the Clinx imaging system (Shanghai, China). Primary antibodies against P4HA1 (1:1000, 12658-1-AP) and β-actin (ACTB, 1:1000, 20536-1-AP) were purchased from Proteintech (Wuhan, China), and against TET2 (1:1000, A5682), FBP1 (1:1000, A11664), P4HA2 (1:1000, A22150), P4HA3 (1:1000, A13767), P4HB (1:1000, A19239) from AbClonal (Wuhan, China). The primary antibody for hypoxia-inducible factor-1α (HIF-1α, GTX127309) was purchased from Gene Tex (Irvine, CA, USA). The primary antibody for Flag (1:1000, MA1-91878) was purchased from Thermo Scientific (USA). Gastrocnemius muscle tissue proteins were extracted using the same procedure followed by homogenization.

For co-immunoprecipitation (co-IP) assays, 2 μg of pCAGEN-Flag-V, pCAGEN-Flag-VN, or pCAGEN-Flag-VC and 2 μg of pCMV-HA-CacyBP/SIP were co-transfected into 293T cells (60 mm well, 12 μl Lipofectamine 2000). After 48 h, the cells were washed with PBS and harvested. The assay was performed according to the manufacturer's instruction for the Pierce™ Co-Immunoprecipitation Kit (26149, Thermo, USA). In brief, the AminoLink Plus Coupling Resin was immobilized by 3.5 μg affinity-purified anti-Flag antibody or mouse IgG as control (MA1-91878, thermo, USA) at 37°C for 2 h. Then, 350 μl ice-cold IP Lysis buffer and protein mixture (FLAG-V and HA-CacyBP/SIP) from one 60 mm plate transfected 293T cells were added to immobilization resin and gently mixed for 2 h at room temperature. After washing, the binding samples were eluted in 60 μl elution buffer via centrifuge at 2,000 g for 2 min. Then, added 5X Sample Buffer to sample to make a 1X final solution and boiled the sample at 100°C for 5 min. Cool the samples to room temperature before applying them to the gel. Anti-HA primary antibodies(26183, thermo, USA) were used for immunoblotting.

The cells were lysed with 1 mL RIPA lysis buffer (P0013B, Beyotime Biotechnology Co., Shanghai, China) in the presence of protease inhibitor in an ice bath for 1 h. The binding of circ_000829 to SRSF1 and that of SRSF1 to SLC39A14 mRNA subtype were detected using the RIP kit (Merck Millipore, Billerica, MA). An aliquot of 10 μg cell extract was collected as input. One portion of 200 μg cell lysate was incubated with flag antibody, and another portion of 200 μg was incubated with isotype control antibody IgG. Cell lysates were incubated with RIP buffer containing magnetic beads MA1-91878 (Thermo Fisher Scientific). The beads were added with RNase inhibitor and incubated overnight in a 4°C refrigerator. Magnetic bead products were incubated at 65°C for 45 min with 117 μL RIP buffer, 15 μL 10% SDS, and 18 μL proteinase K. The isolated RNA was purified and stored at -80°C. The purified RNA product was used for subsequent qPCR detection. Antibodies used in this assay included rabbit anti-Flag (1 : 100, AE063, ABclonal, China) and rabbit antihuman IgG (1 : 100, ab109489, Abcam, serving as a NC). The Protein A/G magnetic beads (HY-K0202, MCE, Sovizzo Vicenza, Italia) used were 100 μL/reaction, and the concentration was 1 mg/mL [21 (link)].

The proteins of transfected GC-1 and GC-2 cells were extracted using RIPA buffer (RIPA, Beyotime, Jiangsu, China) and denatured at 100 °C. The denatured proteins were then separated on 10% sodium dodecyl sulfate-polyacrylamide gels, transferred to a polyvinylidene difluoride membrane, and sealed with 5% skim milk at room temperature for 1 h. The primary antibodies were anti-SLC26A1 (1:1,000, NBP1-84897; Novus, St. Charles, MO, USA) and anti-tubulin antibody (1:1,000, MA1-91878, Thermo Fisher Science, Waltham, MA, USA). The membranes were then incubated with secondary antibody (1:3,000, A0208, Beyotime, Jiangsu, China) for 1 h at room temperature. Finally, a quantitative analysis was performed using a SuperSignal West Femto chemiluminescence substrate detection system (Thermo Fisher Scientific, Waltham, MA, USA).

N. benthamiana leaves were grinded in liquid nitrogen, and 1 g of leaf powder was dissolved in a 2-mL extraction buffer GTEN (10% glycerol, 25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA) freshly mixed with 2% (w/v), polyvinylpolypyrrolidone, 1X protease inhibitor, (Thermo #88666) and 10 mM dithiothreitol, 0.1% (v/v) Tween 20. The cell debris was pelleted by centrifugation and supernatant was collected in a fresh tube (first with 3,000 g for 10 min, and again twice with 21,000 g for 10 min in microcentrifuge at 4 °C). For western blot analysis, anti-FLAG (Thermo MA1-91878) and anti-GFP (Thermo MA5-15256) antibodies were used as the primary, and anti-mouse Alkaline Phosphatase conjugated antibody (Chemicon International #AP308A) was used as the secondary antibody.